Quarantine and surveillance strategies for plant pathogen detection and control

Incursions of plant pests and diseases pose serious threats to food security, agricultural productivity and the natural environment. One of the challenges in confidently delimiting and eradicating incursions is how to choose from an arsenal of surveillance and quarantine approaches in order to best control multiple dispersal pathways. Anthropogenic spread (propagules carried on humans or transported on produce or equipment) can be controlled with quarantine measures, which in turn can vary in intensity. In contrast, environmental spread processes are more difficult to control, but often have a temporal signal (e.g. seasonality) which can introduce both challenges and opportunities for surveillance and control. This leads to complex decisions regarding when, where and how to search. Recent modelling investigations of surveillance performance have optimised the output of simulation models, and found that a risk-weighted randomised search can perform close to optimally. However, exactly how quarantine and surveillance strategies should change to reflect different dispersal modes remains largely unaddressed. Here we develop a spatial simulation model of a plant fungal-pathogen incursion into an agricultural region, and its subsequent surveillance and control. We include structural differences in dispersal via the interplay of biological, environmental and anthropogenic connectivity between host sites (farms). Our objective was to gain broad insights into the relative roles played by different spread modes in propagating an invasion, and how incorporating knowledge of these spread risks may improve approaches to quarantine restrictions and surveillance. We find that broad heuristic rules for quarantine restrictions fail to contain the pathogen due to residual connectivity between sites, but surveillance measures enable early detection and successfully lead to suppression of the pathogen in all farms. Alternative surveillance strategies attain similar levels of performance by incorporating environmental or anthropogenic dispersal risk in the prioritisation of sites. Our model provides the basis to develop essential insights into the effectiveness of different surveillance and quarantine decisions for fungal pathogen control. Parameterised for authentic settings it will aid our understanding of how the extent and resolution of interventions should suitably reflect the spatial structure of dispersal processes.

Short-term plant-decomposer feedbacks in grassland plants

Plant species differ in their effects on ecosystem productivity and it is recognised that these effects are partly due to plant species-specific influences on soil processes. Until recently, however, not much attention was given to the potential role played by soil biota in these species-specific effects. While soil decomposers are responsible for governing the availability of nutrients for plant production, they simultaneously depend on the amount of carbon provided by plants. Litter and rhizodeposition constitute the two basal resources that plants provide to soil decomposer food webs. While it has been shown that both of these can have effects on soil decomposer communities that differ among plant species, the putative significance of these effects for plant nitrogen (N) acquisition is currently understudied. My PhD work aimed at clarifying whether the species-specific influences of three temperate grassland plants on the soil microfood-web, through rhizodeposition and litter, can feed back to plant N uptake. The methods and approach used (15N labelling of plant litter in microcosm experiments) revealed to be an effective combination of tools in studying these feedbacks. Plant effects on soil organisms were shown to differ significantly between plant species and the effects could be followed across several trophic levels. The labelling of litter further permitted the evaluation of plant acquisition of N derived from soil organic matter. The results show that the structure of the soil microfood-web can have a significant role in plant N acquisition when the structure is experimentally manipulated, such as when comparing systems consisting of microbes to those consisting of microbes and their grazers. However, despite this, the results indicate that differences in N uptake from soil organic matter between different plant species are not related to the effects these species exert on the structure of the soil microfood-web. Rather, these differences in N uptake seem to be determined by other species-specific traits of live plants and their litter. My results thus indicate that different resources provided by different plant species may not induce species-specific decomposer feedbacks on plant N uptake from soil organic matter. This further suggests that the species-specific plant effects on soil decomposer communities may not, at least in the short term, have significant consequences on plant production.

The role of MicroRNAs in stress response in the resurrection plant Tripogon loliiformis

Research in this thesis focussed on the improvement of agricultural crops in increasing water use efficiency that impacts global crop productivity. The study identified key genetic regulatory mechanisms that the resurrection plant Tripogon loliiformis utilises to tolerate desiccation. Due to the conserved nature of the pathways involved, this information can be transferred for the enhancement of drought tolerance and water use efficiency in agricultural crops. Specifically this study used high throughput sequencing, microscopy and plant transformation to further the understanding of post-transcriptional regulatory mechanisms. It was shown that T. loliiformis uses microRNAs to regulate pro-survival autophagy pathways to tolerate desiccation.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.

Peroxidases in lignifying xylem of Norway spruce, Scots pine and silver birch

Lignin is a complex plant polymer synthesized through co-operation of multiple intracellular and extracellular enzymes. It is deposited to plant cell walls in cells where additional strength or stiffness are needed, such as in tracheary elements (TEs) in xylem, supporting sclerenchymal tissues and at the sites of wounding. Class III peroxidases (POXs) are secreted plant oxidoreductases with implications in many physiological processes such as the polymerization of lignin and suberin and auxin catabolism. POXs are able to oxidize various substrates in the presence of hydrogen peroxide, including lignin monomers, monolignols, thus enabling the monolignol polymerization to lignin by radical coupling. Trees produce large amounts of lignin in secondary xylem of stems, branches and roots. In this study, POXs of gymnosperm and angiosperm trees were studied in order to find POXs which are able to participate in lignin polymerization in developing secondary xylem i.e. are located at the site of lignin synthesis in tree stems and have the ability to oxidize monolignol substrates. Both in the gymnosperm species, Norway spruce and Scots pine, and in the angiosperm species silver birch the monolignol oxidizing POX activities originating from multiple POX isoforms were present in lignifying secondary xylem in stems during the period of annual growth. Most of the partially purified POXs from Norway spruce and silver birch xylem had highest oxidation rate with coniferyl alcohol, the main monomer in guaiacyl-lignin in conifers. The only exception was the most anionic POX fraction from silver birch, which clearly preferred sinapyl alcohol, the lignin monomer needed in the synthesis of syringyl-guaiacyl lignin in angiosperm trees. Three full-length pox cDNAs px1, px2 and px3 were cloned from the developing xylem of Norway spruce. It was shown that px1 and px2 are expressed in developing tracheids in spruce seedlings, whereas px3 transcripts were not detected suggesting low transcription level in young trees. The amino acid sequences of PX1, PX2 and PX3 were less than 60% identical to each other but showed up to 84% identity to other known POXs. They all begin with predicted N-terminal secretion signal (SS) peptides. PX2 and PX3 contained additional putative vacuolar localization determinants (VSDs) at C-terminus. Transient expression of EGFP-fusions of the SS- and VSD-peptides in tobacco protoplasts showed SS-peptides directed EGFP to secretion in tobacco cells, whereas only the PX2 C-terminal peptide seems to be a functional VSD. According to heterologous expression of px1 in Catharanthus roseus hairy roots, PX1 is a guaicol-oxidizing POX with isoelectric point (pI) approximately 10, similar to monolignol oxidizing POXs in protein extracts from Norway spruce lignifying xylem. Hence, PX1 has characteristics for participation to monolignol dehydrogenation in lignin synthesis, whereas the other two spruce POXs seem to have some other functions. Interesting topics in future include functional characterization of syringyl compound oxidizing POXs and components of POX activity regulation in trees.

Maintaining plant species richness by cattle grazing - mesic semi-natural grasslands as focal habitats

Semi-natural grasslands are the most important agricultural areas for biodiversity. The present study investigates the effects of traditional livestock grazing and mowing on plant species richness, the main emphasis being on cattle grazing in mesic semi-natural grasslands. The two reviews provide a thorough assessment of the multifaceted impacts and importance of grazing and mowing management to plant species richness. It is emphasized that livestock grazing and mowing have partially compensated the suppression of major natural disturbances by humans and mitigated the negative effects of eutrophication. This hypothesis has important consequences for nature conservation: A large proportion of European species originally adapted to natural disturbances may be at present dependent on livestock grazing and / or mowing. Furthermore, grazing and mowing are key management methods to mitigate effects of nutrient-enrichment. The species composition and richness in old (continuously grazed), new (grazing restarting 3-8 years ago) and abandoned (over 10 years) pastures differed consistently across a range of spatial scales, and was intermediate in new pastures compared to old and abandoned pastures. In mesic grasslands most plant species were shown to benefit from cattle grazing. Indicator species of biologically valuable grasslands and rare species were more abundant in grazed than in abandoned grasslands. Steep S-SW-facing slopes are the most suitable sites for many grassland plants and should be prioritized in grassland restoration. The proportion of species trait groups benefiting from grazing was higher in mesic semi-natural grasslands than in dry and wet grasslands. Consequently, species trait responses to grazing and the effectiveness of the natural factors limiting plant growth may be intimately linked High plant species richness of traditionally mowed and grazed areas is explained by numerous factors which operate on different spatial scales. Particularly important for maintaining large scale plant species richness are evolutionary and mitigation factors. Grazing and mowing cause a shift towards the conditions that have occurred during the evolutionary history of European plant species by modifying key ecological factors (nutrients, pH and light). The results of this Dissertation suggest that restoration of semi-natural grasslands by private farmers is potentially a useful method to manage biodiversity in the agricultural landscape. However, the quality of management is commonly improper, particularly due to financial constraints. For enhanced success of restoration, management regulations in the agri-environment scheme need to be defined more explicitly and the scheme should be revised to encourage management of biodiversity.

The plant Banjauri (Vicoa indica) exhibits antifertility activity in adult female bonnet monkeys (Macaca radiata)

The antifertility activity of the plant Vicoa indica was tested in proven fertile bonnet monkeys. The dry powder of the whole plant was fed to the cycling monkeys on day 1 to 14 of menstrual cycle or day 9 to 14 of cycle or on day 2 to 5 after delivery and the fertility was evaluated in the following cycle in cycle fed monkey or after weaning the young one in the post-partum fed monkeys. Results indicated that while feeding in the post-partum monkeys did not confer any protection against pregnancy feeding during day 1 to 14 of cycle, protected from pregnancy. The monkeys did not become pregnant even after exposure to the proven fertile male monkeys for 13 ovulatory cycles while all the vehicle fed monkeys became pregnant within 3 cycles.

Plant regeneration from protoplasts of Capsicum annuum L cv. California Wonder

Plant regeneration from mesophyll protoplasts of pepper, Capsicum annuum L. cv. California Wonder has been demonstrated via shoot organogenesis, Protoplasts isolated from fully expanded leaves of 3-week-old axenic shoots when cultured in TM medium supplemented with 1 mgl(-1) NAA, 1 mgl(-1) 2, 4-D, 0.5 mgl(-1) BAP (CM 1) resulted in divisions with a frequency ranging from 20-25%. Antioxidant ascorbic acid and polyvinylpyrrolidone (PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed in TM medium containing 2 mgl(-1) NAA and 0.5 mgl(-1) BAP (CM II) and MS gelled medium containing 2 mgl(-1) NAA and 0.5 mgl(-1) BAP (CM III), respectively, Regeneration of plantlets was possible via caulogenesis, Microshoots, 2-5 per callus appeared on MS gelled medium enriched with 0.5 mgl(-1) IAA, 2 mgl(-1) GA and 10 mgl(-1) BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mgl(-1) NAA and 0.5 mgl(-1) BAP, Protoplasts isolated from cotyledons failed to divide and degenerated eventually.

Characterization of Somatic Embryogenesis in Sandalwood (Santalum album L.)

In the synchronous embryogenesis system of sandalwood developed in our laboratory, we observed that the early events of differentiation from freshly induced callus (stage 0) are accomplished in three distinct stages viz., preglobular masses (stage 1), globular embryos (stage 2), and bipolar embryos (stage 3). Transition from stage 0 to 1 was accomplished using 2,4-D and involves a stage specific appearance of two polypeptides of 15 and 30 kDa molecular weight. A 24 kDa polypeptide that was detected as a marked band in extracts of primary callus was not detected in stages 1, 2, and 3. Further, the tissue level of a 50 kDa glycoprotein decreased during transition from stage 2 to stage 3. However, the levels of glycoproteins in the medium were markedly higher in stage 0 cultures compared to those in stage 1. The activities of a protein kinase, glycosidase, and xylanase increased markedly with progressing embryogenesis. Our observations suggest that in addition to being controlled at the level of stage-specific gene expression, somatic embryogenesis in sandalwood is also regulated at the level of controls on cell wall flexibility and posttranslational changes in the pool of preexisting proteins.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H2O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.

Trehalose accumulation triggers autophagy during plant desiccation

Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis , by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops.