Gene transfer to plants by diverse species of bacteria

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology1. However, the complexity of the patent landscape2 has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile ‘open source’ platform for plant biotechnology, may lead to new uses of natural bacteria– plant interactions to achieve plant transformation.

Bioavailability of soil organic carbon and Fe as influenced by forestry practices in a subtropical coastal catchment

Potential impacts of plantation forestry practices on soil organic carbon and Fe available to microorganisms were investigated in a subtropical coastal catchment. The impacts of harvesting or replanting were largely limited to the soil top layer (0–10 cm depth). The thirty-year-old Pinus plantation showed low soil moisture content (Wc) and relatively high levels of soil total organic carbon (TOC). Harvesting and replanting increased soil Wc but reduced TOC levels. Mean dissolved organic carbon (DOC) and microbial biomass carbon (MBC) increased in harvested or replanted soils, but such changes were not statistically significant (P > 0.05). Total dithionite-citrate and aqua regia-extractable Fe did not respond to forestry practices, but acid ammonium oxalate and pyrophosphate-extractable, bioavailable Fe decreased markedly after harvesting or replanting. Numbers of heterotrophic bacteria were significantly correlated with DOC levels (P < 0.05), whereas Fe-reducing bacteria and S-bacteria detected using laboratory cultivation techniques did not show strong correlation with either soil DOC or Fe content.

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were colleted during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples colleted during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both, C. jejuni mapA and Salmonella invA genes were detected in 10% and 10% of samples, respectively collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 102 to 4.3 × 102 genomic copies per 500 ml of water G. lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.

Characterisation of the tritrophic interactions between tobacco yellow dwarf virus, its vector and host-plants

Tobacco yellow dwarf virus (TbYDV, family Geminiviridae, genus Mastrevirus) is an economically important pathogen causing summer death and yellow dwarf disease in bean (Phaseolus vulgaris L.) and tobacco (Nicotiana tabacum L.), respectively. Prior to the commencement of this project, little was known about the epidemiology of TbYDV, its vector and host-plant range. As a result, disease control strategies have been restricted to regular poorly timed insecticide applications which are largely ineffective, environmentally hazardous and expensive. In an effort to address this problem, this PhD project was carried out in order to better understand the epidemiology of TbYDV, to identify its host-plant and vectors as well as to characterise the population dynamics and feeding physiology of the main insect vector and other possible vectors. The host-plants and possible leafhopper vectors of TbYDV were assessed over three consecutive growing seasons at seven field sites in the Ovens Valley, Northeastern Victoria, in commercial tobacco and bean growing properties. Leafhoppers and plants were collected and tested for the presence of TbYDV by PCR. Using sweep nets, twenty-three leafhopper species were identified at the seven sites with Orosius orientalis the predominant leafhopper. Of the 23 leafhopper species screened for TbYDV, only Orosius orientalis and Anzygina zealandica tested positive. Forty-two different plant species were also identified at the seven sites and tested. Of these, TbYDV was only detected in four dicotyledonous species, Amaranthus retroflexus, Phaseolus vulgaris, Nicotiana tabacum and Raphanus raphanistrum. Using a quadrat survey, the temporal distribution and diversity of vegetation at four of the field sites was monitored in order to assess the presence of, and changes in, potential host-plants for the leafhopper vector(s) and the virus. These surveys showed that plant composition and the climatic conditions at each site were the major influences on vector numbers, virus presence and the subsequent occurrence of tobacco yellow dwarf and bean summer death diseases. Forty-two plant species were identified from all sites and it was found that sites with the lowest incidence of disease had the highest proportion of monocotyledonous plants that are non hosts for both vector and the virus. In contrast, the sites with the highest disease incidence had more host-plant species for both vector and virus, and experienced higher temperatures and less rainfall. It is likely that these climatic conditions forced the leafhopper to move into the irrigated commercial tobacco and bean crop resulting in disease. In an attempt to understand leafhopper species diversity and abundance, in and around the field borders of commercially grown tobacco crops, leafhoppers were collected from four field sites using three different sampling techniques, namely pan trap, sticky trap and sweep net. Over 51000 leafhopper samples were collected, which comprised 57 species from 11 subfamilies and 19 tribes. Twentythree leafhopper species were recorded for the first time in Victoria in addition to several economically important pest species of crops other than tobacco and bean. The highest number and greatest diversity of leafhoppers were collected in yellow pan traps follow by sticky trap and sweep nets. Orosius orientalis was found to be the most abundant leafhopper collected from all sites with greatest numbers of this leafhopper also caught using the yellow pan trap. Using the three sampling methods mentioned above, the seasonal distribution and population dynamics of O. orientalis was studied at four field sites over three successive growing seasons. The population dynamics of the leafhopper was characterised by trimodal peaks of activity, occurring in the spring and summer months. Although O. orientalis was present in large numbers early in the growing season (September-October), TbYDV was only detected in these leafhoppers between late November and the end of January. The peak in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and was also associated with warmer temperatures and lower rainfall. To understand the feeding requirements of Orosius orientalis and to enable screening of potential control agents, a chemically-defined artificial diet (designated PT-07) and feeding system was developed. This novel diet formulation allowed survival for O. orientalis for up to 46 days including complete development from first instar through to adulthood. The effect of three selected plant derived proteins, cowpea trypsin inhibitor (CpTi), Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), on leafhopper survival and development was assessed. Both GNA and WGA were shown to reduce leafhopper survival and development significantly when incorporated at a 0.1% (w/v) concentration. In contrast, CpTi at the same concentration did not exhibit significant antimetabolic properties. Based on these results, GNA and WGA are potentially useful antimetabolic agents for expression in genetically modified crops to improve the management of O. orientalis, TbYDV and the other pathogens it vectors. Finally, an electrical penetration graph (EPG) was used to study the feeding behaviour of O. orientalis to provide insights into TbYDV acquisition and transmission. Waveforms representing different feeding activity were acquired by EPG from adult O. orientalis feeding on two plant species, Phaseolus vulgaris and Nicotiana tabacum and a simple sucrose-based artificial diet. Five waveforms (designated O1-O5) were observed when O. orientalis fed on P. vulgaris, while only four (O1-O4) and three (O1-O3) waveforms were observed during feeding on N. tabacum and the artificial diet, respectively. The mean duration of each waveform and the waveform type differed markedly depending on the food source. This is the first detailed study on the tritrophic interactions between TbYDV, its leafhopper vector, O. orientalis, and host-plants. The results of this research have provided important fundamental information which can be used to develop more effective control strategies not only for O. orientalis, but also for TbYDV and other pathogens vectored by the leafhopper.

Design and implementation of sensor networks for the observation and research of harmful algal blooms in southern California coastal waters

Harmful Algal Blooms (HABs) have become an important environmental concern along the western coast of the United States. Toxic and noxious blooms adversely impact the economies of coastal communities in the region, pose risks to human health, and cause mortality events that have resulted in the deaths of thousands of fish, marine mammals and seabirds. One goal of field-based research efforts on this topic is the development of predictive models of HABs that would enable rapid response, mitigation and ultimately prevention of these events. In turn, these objectives are predicated on understanding the environmental conditions that stimulate these transient phenomena. An embedded sensor network (Fig. 1), under development in the San Pedro Shelf region off the Southern California coast, is providing tools for acquiring chemical, physical and biological data at high temporal and spatial resolution to help document the emergence and persistence of HAB events, supporting the design and testing of predictive models, and providing contextual information for experimental studies designed to reveal the environmental conditions promoting HABs. The sensor platforms contained within this network include pier-based sensor arrays, ocean moorings, HF radar stations, along with mobile sensor nodes in the form of surface and subsurface autonomous vehicles. FreewaveTM radio modems facilitate network communication and form a minimally-intrusive, wireless communication infrastructure throughout the Southern California coastal region, allowing rapid and cost-effective data transfer. An emerging focus of this project is the incorporation of a predictive ocean model that assimilates near-real time, in situ data from deployed Autonomous Underwater Vehicles (AUVs). The model then assimilates the data to increase the skill of both nowcasts and forecasts, thus providing insight into bloom initiation as well as the movement of blooms or other oceanic features of interest (e.g., thermoclines, fronts, river discharge, etc.). From these predictions, deployed mobile sensors can be tasked to track a designated feature. This focus has led to the creation of a technology chain in which algorithms are being implemented for the innovative trajectory design for AUVs. Such intelligent mission planning is required to maneuver a vehicle to precise depths and locations that are the sites of active blooms, or physical/chemical features that might be sources of bloom initiation or persistence. The embedded network yields high-resolution, temporal and spatial measurements of pertinent environmental parameters and resulting biology (see Fig. 1). Supplementing this with ocean current information and remotely sensed imagery and meteorological data, we obtain a comprehensive foundation for developing a fundamental understanding of HAB events. This then directs labor- intensive and costly sampling efforts and analyses. Additionally, we provide coastal municipalities, managers and state agencies with detailed information to aid their efforts in providing responsible environmental stewardship of their coastal waters.

Implementation of an embedded sensor network for the coordination of Slocum gliders for coastal monitoring and observation

Autonomous Underwater Vehicles (AUVs) are revolutionizing oceanography through their versatility, autonomy and endurance. However, they are still an underutilized technology. For coastal operations, the ability to track a certain feature is of interest to ocean scientists. Adaptive and predictive path planning requires frequent communication with significant data transfer. Currently, most AUVs rely on satellite phones as their primary communication. This communication protocol is expensive and slow. To reduce communication costs and provide adequate data transfer rates, we present a hardware modification along with a software system that provides an alternative robust disruption- tolerant communications framework enabling cost-effective glider operation in coastal regions. The framework is specifically designed to address multi-sensor deployments. We provide a system overview and present testing and coverage data for the network. Additionally, we include an application of ocean-model driven trajectory design, which can benefit from the use of this network and communication system. Simulation and implementation results are presented for single and multiple vehicle deployments. The presented combination of infrastructure, software development and deployment experience brings us closer to the goal of providing a reliable and cost-effective data transfer framework to enable real-time, optimal trajectory design, based on ocean model predictions, to gather in situ measurements of interesting and evolving ocean features and phenomena.

Networked robotic sensor platform deployments for use in coastal environmental assessment in southern California

Mobile sensor platforms such as Autonomous Underwater Vehicles (AUVs) and robotic surface vessels, combined with static moored sensors compose a diverse sensor network that is able to provide macroscopic environmental analysis tool for ocean researchers. Working as a cohesive networked unit, the static buoys are always online, and provide insight as to the time and locations where a federated, mobile robot team should be deployed to effectively perform large scale spatiotemporal sampling on demand. Such a system can provide pertinent in situ measurements to marine biologists whom can then advise policy makers on critical environmental issues. This poster presents recent field deployment activity of AUVs demonstrating the effectiveness of our embedded communication network infrastructure throughout southern California coastal waters. We also report on progress towards real-time, web-streaming data from the multiple sampling locations and mobile sensor platforms. Static monitoring sites included in this presentation detail the network nodes positioned at Redondo Beach and Marina Del Ray. One of the deployed mobile sensors highlighted here are autonomous Slocum gliders. These nodes operate in the open ocean for periods as long as one month. The gliders are connected to the network via a Freewave radio modem network composed of multiple coastal base-stations. This increases the efficiency of deployment missions by reducing operational expenses via reduced reliability on satellite phones for communication, as well as increasing the rate and amount of data that can be transferred. Another mobile sensor platform presented in this study are the autonomous robotic boats. These platforms are utilized for harbor and littoral zone studies, and are capable of performing multi-robot coordination while observing known communication constraints. All of these pieces fit together to present an overview of ongoing collaborative work to develop an autonomous, region-wide, coastal environmental observation and monitoring sensor network.