Summary PPH 31306 Seed Science & Technology (1) PDF

Romy Steenkamer Seed science and technology PPH 31306 Lecture 1 Introduction chapter 1 Importance of seeds Seeds are a major food source: ~ 90% of the annual worldwide seed production contributes to 50% of the energy intake. The Netherla...

Romy Steenkamer Seed science and technology PPH 31306 Lecture 1 Introduction chapter 1 Importance of seeds Seeds are a major food source: ~ 90% of the annual worldwide seed production contributes to 50% of the energy intake. The Netherlands are number 1 seed exporting country. Seeds preserve natural environments (biodiversity): soil seed banks determine next generation vegetation. Some species are endangered by climate change. In gene banks seeds are safeguarded. The seed bank Svalbard in Norway has copies of all accessions from other seed banks worldwide. Here seeds are stored under controlled conditions in the permafrost and can be used to replant destroyed areas. Lecture 2 What is a seed? Seeds form the next generation of a plant, containing a complete new generation (the embryo), as well as other supportive (endosperm, perisperm or cotyledons for angiosperms and megagametophyte for gymnosperms) and protective (seed coat) structures. Seeds are products of fertilization between pollen and egg. Pollen and egg are produced by seedplants: in the flowers of angiosperms and in the cones of gymnosperms. Angiosperms can be either monocots or dicots Seed structures: the different tissues of a seed Embryo: next generation of the plant, formed by fertilization of the egg cell nucleus in the embryo by one of the male pollen tube nuclei, that is ♀♂ Endosperm: nutritional tissue, arises from the fusion of two polar nuclei of the central cell in the embryo sac with the other pollen tube nucleus, thus ♀♀♂ Seed coat: protective tissue, also called the testa, derived from the integument(s) around the ovule and thus ♀♀. Funiculus: the umbilical cord, structure that joined the seed to its parent plant Hilum: scar marking the point at which the seed was joined to the funiculus Micropyle: a small depression at one end of the hilum (not present in all seed coats)  Romy Steenkamer Different seeds have different seed structures True seeds: do not contain the maternal pericarp tissue: legumes, cotton, tomato, squashes, coffee bean produces true seeds. Fruits are seeds without a (complete) seed coat but with a pericarp: cereals, lettuce, sunflower, nuts, buttercup, anemone.  Romy Steenkamer Seed reserves -Storage reserves: Carbohydrates, oils and proteins -Others: Alkaloids, lectins, proteinase inhibitors, phytin and raffinose family oligosaccharides (RFOs) Average percentages storage composition and location differ between different crop species. Protein Oil Carbohydrate Major storage location Cereals 11 5 84 Endosperm Legumes 29 8 53 Cotyledons Agricultural revolution(s) Seeds are (at) the basis of the agricultural system. (Starting material & end product). 1. During the First Agricultural Revolution/ Neolithic Revolution (circa 10 000 BC), the prehistoric transition from hunting and gathering to settled agricultural took place. 2. The Second Agricultural Revolution/ British Agricultural Revolution (17th – 19th century) involved the introduction of new crop rotation techniques and selective breeding of livestock, resulting in a marked increase in agricultural production. 3. The Third Agricultural Revolution/ Green Revolution (1930 - 1960) concerns an increase in agricultural production as a consequence of irrigation, specialized seeds, machinery, fertilizers and pesticides. Especially took place in the developing world. Domestication: the initiation of the process of evolutionary divergence from wild ancestral species. For examples wheat, maize and rice. Diversification: the subsequent evolution of new varieties, including greater improvement in yield, adaptation or quality in crop species. Commonly observed traits in crops after domestication (1) and diversification 2-4): Stage 1 Stage 2 Stage 3 Stage 4 Larger seeds More seeds Reduced Increased yield vernalization Resource allocation Pigment Reduced photoperiod Increased abiotic variation sensitivity stress tolerance Thinner seed coat, Increased seed Modified hormone Increased biotic increased seed-softening size variation sensitivity stress tolerance and ornamentation Inflorescence architecture Flavour change Synchronized Improved eating (shape, number, flowering time quality determinacy ) Increased yield potential Change in Shortened or and productivity starch content extended life cycle Loss of dormancy Reduced Dwarfism germination inhibition Determinate growth Non-shattering seeds  Romy Steenkamer In different crops, different natural mutations in the same genes were selected for the case of non-shattering and plant architecture. Seeds as populations Dormancy release or germination of seeds are binary responses: either a seed germinates or not. Relation between such responses and abiotic factors are represented in a Sigmoid curve like the one below. This behaviour is called ‘Threshold’ behaviour in which a certain abiotic factor needs to be exceed before a process occurs. As individual seeds vary in their sensitivity to temperature, light, moisture, often over a logarithmic concentration range the Sigmoid curve represents the relationship between the response (germination) percentage over time. Population-based-threshold (PBT) models are useful in describing the duality of individual diversity (each seed relies on its own resources to persist, germinate and grow into a seedling) and population-wide-behaviour (the percentage of seeds in a population that is in an particular state). As thresholds may be different for individual seeds, , population based models capture variability by averaging. ΘX = (X – Xb(i))ti ΘX = the time constant for responses to factor X X = the dosage level of factor X Xb(i) = the sensitivity threshold distribution of the population for a given phenotype or response Ti = the time at which fraction i of the population exhibits the phenotype or response due to factor level X  Romy Steenkamer All biological systems may function as population. Lectures 3 & 4 Seed maturation chapter 2 Spermatophytes are seed producing plants; thus gymnosperms and angiosperms (monocots or dicots). Seeds are formed by double fertilization. 2 sperm cells enter the pollen tube, one fertilizes the egg cell, resulting in the embryo and the other fertilizes the central cell (with 2 polar nuclei) to form the endosperm. Roles of the seed parts Embryo ♂♀: contains all the parts necessary to form a seedling Endosperm ♂♀♀: provides nutrition to the embryo Endospermic seeds: rice, maize, coffee, Arabidopsis Non-endospermic seeds (cotyledonary): peas, common bean, onion, vanilla Seed coat/ testa ♀♀: - Waterproofing (waxes, fats/ oils) - Water retention (mucilages) - Protection (structure, chemicals) - Impermeability (O2, water) - Impose (induce) dormancy - Dispersal (outgrowths, attractants) - Conduit for assimilates during development  Romy Steenkamer Seed development: the formation of the embryo and other seed tissues through highly ordered patterns of cell division and differentiation. (Plant → flower → fertilized egg cell → (globular) embryo → heart-shaped embryo → torpedo embryo → maturation → dormant seed → seedling Orthodox seeds: survive desiccation Intermediate seeds: not tolerant to drying as much as orthodox seeds and sensitive to low temperatures. Cannot be stored under conventional seed bank conditions, poor longevity (only a few months) Recalcitrant seeds: do not go undergo and thus not survive drying at the end of maturation and are often maintained as living plant collections. (Citrus, coffee, cocoa, mango, avocado). Primary found in evergreen forests with optimal conditions for germination. Anthesis is the onset of flowering  Romy Steenkamer The 3 phases of seed development and accompanying physiological events: Fresh weight and water content increase linearly during embryogenesis due to cell divisions and imbibition, during maturation only dry weight proceeds to increase as the water content levels of during maturation drying (! Does not occur in all species). During the final drying phase only dry weight increases due to storage of reserves. During embryogenesis or morphogenesis the polarity of the embryo is determined and tissues are formed. Any mutations during this phase affects normal seedling development.  Romy Steenkamer Throughout maturation seed filling, reserve accumulation and moisture content changes occur. The reserve accumulation during seed maturation determines the nutritional value of seeds. After completion of this phase the nutritional value is fixed. Seed quality traits become visible and are determined during the maturation phase, examples can be morphological as well as physiological. Example dynamics physiology and acquisition quality traits B. water content decreases, dry weight increases C. simultaneously desiccation tolerance and germination capacity increase D ability to germinate under stressful conditions increases  Romy Steenkamer Late maturation involves the completion of trait acquisition and drying. Relative lengths of this phase differ across species. At the point of maturity: seeds have become fully functional, some species acquire this post-harvest (need to be stored in the refrigerator). Hormonal regulation of seed development Abscisic acid and gibberellins play a major role during seed development, maturation and germination. Auxin, ethylene and cytokinin are essential, as well. Brassinosteroids, salicylic acid, strigolactones and karrikins are important post- development/ germination. Roles of ABA in angiosperms Germination Dormancy Biotic stress Abiotic stress Fruit ripening Senescence Abscission  Romy Steenkamer 2 peaks in ABA levels during seed development can be observed. The first one corresponds to the acquisition of desiccation tolerance and the second to dormancy. Roles of GA in angiosperms Flower development Embryo development Trichrome development Shoot apical meristem development Gibberellins constitute of a group of several molecules. The different forms have different functions. Hormones need to be metabolized as well as Biosynthesis Catabolism degraded. The table right shows the involved genes. ABA NCED CYP707A1 GA GA20ox, GA3ox GA2ox Transcriptional regulation ABA and GA metabolism and signalling are intimately connected in seeds. The ABA:GA balance determines seed dormancy and germination. The four major regulators of seed development are LAFL transcription factors: Wild-type Lec1 Lec2 Fus3 Abi3 Desiccation tolerant Intolerant Partially Intolerant Intolerant tolerance intolerant Seed protein normal abnormal abnormal abnormal abnormal gene expression Shoot apex not activated activated Activated activated activated Activation normal premature No data (not No data (not premature of post detected) detected) germinative gene expression ABA sensitive sensitive sensitive sensitive Reduced sensitivity Dormancy dormant reduced reduced reduced reduced  Romy Steenkamer Abi3-6 strong mutants not only have reduced desiccation tolerance and dormancy but also produce green seeds, as a consequence of defects in chlorophyll degradation during seed maturation. The LAFL transcriptional network and hormones together affect gene expression involved with the entire seed development program The VAL (VIVIPAROUS1/ABI3-LIKE1/2/3) is a sister-clade of the LAFL transcription factors. It represses the LAFL network during seed germination. In conjunction the LAFL and VAL transcription factor networks regulate the seed to seedling transition. Pre-harvest sprouting absence or lack of sensitivity to ABA may lead to viviparous germination (germination on the mother plant, no developmental arrest after completion of embryogenesis). - Precocious (premature) germination of grains prior to harvest - Occurs when cool moist conditions persist close to the time of harvest - Causes economic losses in grain quality for baking - Relevant in cultivars with low grain dormancy (less dormant is more susceptible to PHS)  Romy Steenkamer Strategies to increase dormancy and prevent PHS - upregulate genes upstream ABA - downregulate or knock-out genes downstream ABA - downregulate gene upstream GA - Inhibit expression GA Physiological and cellular markers of seed maturation Many plants have an extended flowering period, which makes it difficult to tell when most of the seeds are mature. A single harvest can contain seeds from different maturity levels. Seed maturation analyses are species-specific Water content Chlorophyll degradation Accumulation of Raffinose Family Oligosaccharides Genetic or molecular markers are useful to identify the maturity level of a seed batch - content of hormones - genetic markers: transcription factors, enzymes, protective proteins LEAs & HSPs Lectures 5 & 6 Seed survival chapter 8 Seed survival in the environment Seed development and maturation prepare seeds for: Dispersal (maternal structures, fruits) Dry periods (desiccation tolerance) Long-term survival (longevity) Reside in the soil for long periods (dormancy cycling) Germination (reserve food accumulation) A cross-talk between important seed maturation events exist  Romy Steenkamer Desiccation tolerance: the ability to survive the loss of almost cellular water without irreversible damages. Important for biodiversity conservation, agriculture, economy and food security. Extremophiles show an enormous desiccation tolerance. Examples can be found across kingdoms: yeast, nematodes, rotifers, tardigrades, unicellular organisms and plants. In plants both reproductive (in spores, seeds) as well as vegetative desiccation tolerance (in cells, leaves) can be found. Drought tolerance ≠ desiccation tolerance Drought tolerant plants either evade, avoid or tolerate water loss. In the case of tolerating water loss, drought tolerant plants are desiccation sensitive: only small amount of water, 2%- 50% of their relative water content for short periods of time (minutes-hours) can be withstand. Desiccation tolerant plants possess the ability to lose 95% of their relative water content for prolonged periods (months-years) without dying. Drying phases - Phase I: loss of free water - Phase II: loss of hydrogen bonds bounded water - Phase III: loss of tightly bound water An sorption isotherm describes the relationship between equilibrium relative humidity and moisture content at a constant temperature and pressure. Desiccation tolerance strategies - Accumulation of soluble sugars (e.g. RFOs) - Accumulation protective proteins (e.g. LEA and HSP) - Accumulation of osmolytes  Romy Steenkamer The general mechanism of desiccation tolerance in seeds in protecting against cellular damages caused by drying. Seeds adopt a glassy state: an amorphous solid-like state that retains the physical properties of a fluid in which water is replaced by sugars like RFOs, LEA proteins. Raffinose family oligosaccharides (RFOs) Soluble sugars Thought to be important for the ‘fluid-viscous-glassy’ state transition Contribute to phase separations, as these molecules replace the scarce water other molecules can keep their original shape Accumulate during seed maturation and/ or drying phases Late Embryogenesis Abundant (LEA) proteins Highly hydrophilic and heat-stable Intrinsically disordered proteins Become folded and functional upon heating Accumulate in seed maturation Protect against: - membrane breakage - Protein denaturation - Ions and ROS accumulation  Romy Steenkamer Early LEAs are more important in desiccation tolerance Late LEAs are more important in longevity (early and late during maturation) Temporal separation between transcription and translation Knock out of LEAs cause problems in: - Maturation - Acquisition of desiccation tolerance - Longevity/ storability Overexpression of LEAs can improve: - Plant drought tolerance - Seedling growth under osmotic stress - Plant salt stress tolerance Both LEA as well as RFO are regulated via ABA → ABAI3 Loss and re-induction of desiccation tolerance Desiccation tolerance is lost during germination, usually when the radicle protrudes from the surrounding tissues. The seed-to-seedling transition is irreversible and marks a point of no return. It is possible, however, to re-induce desiccation tolerance in germinating seeds using polyethylene glycol (PEG). To do so, ABA is necessary because it regulates LEA and RFO expression. Longevity: The period in which (seeds) remain viable in the dry state. Natural deterioration processes (increasing DNA damage, protein adducts and lipid peroxidation) can be enhanced during storage (by oxygen, temperature, UV light), leading to a faster seed aging and, consequently, decreasing seed longevity. Mechanisms of seed longevity  Romy Steenkamer Environmental conditions during the maturation phase of seed development influence gene expression of hormones and transcription factors. These in turn regulate several longevity mechanisms: - Stabilization and glassy state - Antioxidants - Chaperones - Repair mechanisms - Seed coat Reactive oxygen species (ROS) can be formed in seeds by: - Enzymatic activity during late maturation - Uncontrolled slowing down of energy metabolism during drying - Respiration (during maturation, drying and storage) - Exposure to UV High ROS levels have negative effects: - Changing membrane permeability & organelle functioning - Affecting DNA replication and cell division resulting in oxidative damage to DNA and RNA - Altered mRNA translation producing (mis)folded proteins and affecting enzyme functioning Antioxidants help to convert (enzymatic antioxidants) or scavenge (non-enzymatic antioxidants) ROS and increase longevity by inhibiting the loss of electrons (oxidation). Lower seed coat permeability contributes to seed longevity: hydrophobic compounds on the seed coat (lignin, suberin) can reduce the permeability to moisture and oxygen during storage. Both ABA as well as Auxin contribute to seed longevity, dormancy and desiccation tolerance via transcription factorABI3.  Romy Steenkamer Important points to consider during seed storage longevity analysis Before storage During storage After storage Wet aging Drying treatment Water content harvested Temperature, RH and seed Preimbibition seeds water content, changes in FW treatment at 100% Further drying treatment Hermetically sealed or open RH before Length pre-equilibration air storage germination treatment at set RH and Dry aging testing temperature Temperature, RH, seed water content Hermitically sealed open air storage Desiccation tolerance is acquired before and necessary for seed longevity. Organisms first need to survive drought (and thus have desiccation tolerance) before longevity is acquired. To identify which regulators are specific for and which are shared between these seed quality traits the a co-expression network can be constructed: → RNA seq of seeds from different days after pollination to find expressed genes at certain DAP → CO-expression analysis to determine relationships between expressed genes → Network construction: visualization of the relationships between gene groups → Module definition: identification of strong connections between gene groups → Biological meaning: relationships between modules & phenotypes could result in the identification of biological processes and regulators Consequent validation of the co-expression network to make sure it is correct and can point to the true regulators may involve: 1. Identification of major hubs (= highly connected genes) 2. Gene knock-out and overexpression: study if gene is necessary for phenotype 3. Mutant gene complementation: study if the gene is sufficient to rescue the phenotype 4. RNA-seq or RT-qPCR of neighbouring genes: validate if KO affects expression of co- expressed genes and identify biological processes that are affected in the mutant 5. KO or OE of homologs in other species 6. Gain more knowledge in the regulation Conclusions Seeds can only be long-lived in the dry state if they are desiccation tolerant Desiccation tolerance and longevity are differentially regulated by their specific gene modules Desiccation tolerant seeds have a core protection mechanism including specific proteins (LEAs) and sugars Maturation environment and storage conditions are major determinants of seed longevity  Romy Steenkamer Gene regulatory network analyses can help on identifying major regulators of seed survival in the environment The death of dry-stored seeds is predictable and rules of thumb for seed storage exist James’ rule: Temp (ºC) + RH (%) < 60%to obtain a reasonable shelf life. Harrington’s rule: Seed longevity will ~ double for each 5.6 ºC decrease in temperature and each 1% decrease in seed moisture content for temperatures between 0-40 ºC and moisture content between 5 and 14%. Seeds in the soil - endure variable conditions: moisture, temperature, predation, decay affect soil seed banks. - persist until they either germinate or die due to aging, predation or decay - survival chance depends on dormancy type, longevity, defence mechanisms and exposure to biotic and abiotic factors Seed ecology: The complexity of seed survival and seedling establishment in the soil can be used to predict the effects of climate change on plant communities.  Romy Steenkamer Summary - Seed survival in the soil is generally shorter than in dry storage - Many variables and their mutual interactions, determine persistence of seeds in the soil - Seed ecology may explain the settlement, maintenance and decay of plant communities under varying conditions - Recalcitrant seeds do not undergo maturation drying, are thus sensitive to desiccation and have a short longevity - Intermediate seeds can tolerate some drying, but are sensitive to low storage temperature - Both recalcitrant and intermediate seeds lack the activation of important processes related to desiccation tolerance and longevity during maturation. (LEA proteins by ABI3) Lectures 7 & 8 Seed dormancy chapter 6 Seed dormancy is the temporary failure of a (viable) seed to complete germination under favourable conditions. Dormancy provides a strategy for seeds to spread germination in time in order to reduce the risk of premature death in an unfavourable environment; - Reducing competition as different seeds have different levels of dormancy - Distribution of germination over time - Distribution of germination over space when dormant seeds are dispersed over long distances. Embryo dormancy: the properties of the embryo are of principal importance. This is physiological dormancy: it is reversible, widespread and central to the phenomenon of seasonal dormancy cycling of seeds in the soil. Coat-imposed dormancy: properties of the covering tissues are determinative and thus irreversible. This is physical dormancy. Primary dormancy: seeds that are shed from the parent plant in a dormant state Secondary dormancy: acquired by seeds in the soil if germination conditions are unfavourable or if seed germination is inhibited by other means (any stresses). Relieving dormancy - After-ripening: variable periods of dry storage to break dormancy in many species. High summer temperatures break dormancy in winter annuals, whereas cold-stratification is effective in breaking dormancy of many summer annuals. The rate of after-ripening decrease can vary depending on environmental conditions during seed maturation (lower temperatures during seed development result in more dormant seed), seed storage and germination conditions. After-ripening may be prevented at higher seed moisture content and viability may be lost as well (>20-40%), at higher water contents dormancy is maintained or secondary dormancy may be induced. If seeds become too dry, on the other hand, after-ripening is delayed or  Romy Steenkamer prevented. Besides the influence of water, after-ripening is delayed when oxygen tensions are low. A decrease in ABA content and sensitivity are associated with after-ripening. Molecular mechanisms that decrease dormancy involve various chemical changes in dry seed: - mRNA’s of dormancy-associated genes, including DOG1, decrease - several proteins accumulated, possibly due to change in mobility or by chemical modifications Dormancy may be broken as well by temperature fluctuations. The combination of a certain minimum temperature and maximum temperature is then essential. - Light: Almost all light-requiring seeds have coat-imposed dormancy. Light-sensitivity in many species is enhanced by chilling, various temperature alternations and temperature shifts also interact with light. White light breaks dormancy, wavelengths in the orange/red region are the most effective (660 nm). Inhibitory parts of the spectrum can be found at the far-red spectrum (730 nm). Photoreversibility: the wavelengths 660 and 730 nm can reverse each other’s effect. Dormant seeds mostly possess the unactive phytochrome (Pr) upon irradiation of red light it is converted to its active version Pfr, which can break dormancy. Upon irradiation with light of 730 nm P fr will be reconverted to Pr. All phytochrome molecules within one plant will never be all in the same state: it is the mixture of phytochrome states that together are important for germination. The mixture and overlap in absorptionspectra results in the photo-equilibrium: Φ=Pfr / Ptot a) Incandescent light is relatively rich in far-red light wavelengths compared to b) fluorescent light, which is relatively rich in red wavelengths. Seeds maturing in fluorescent light lose their dormancy faster as they can accumulate active phytochrome necessary for germination.  Romy Steenkamer Hormonal regulation No ABA: negative regulator PP2C binds to positive regulator SnRK2. Upon ABA perception: PP2C is bound to the ABA receptor, the now free SnRK2 will be phosphorylated and activates genes that induce dormancy. No GA: RGL2 protein suppresses expression of GA-inducible genes. Upon GA perception: The protein GID1 binds to GA, and interacts with RGL2 (a DELLA protein). This triggers its ubiquitination. The ubiquitinated RGL2 is recognized by the 26S proteasome and degraded. The repression of GA-inducible genes is removed and dormancy is released.. Hormonal crosstalk  Romy Steenkamer Major dormancy regulator: Delay Of Germination1 A high correlation of DOG1 expression with dormancy levels during seed maturation exists. DOG1 acts as a temperature sensor during seed maturation. The level of DOG1 transcripts are higher under unfavourable conditions, which delays radicle emergence. A lack of correlation between DOG1 expression and dormancy levels were found during imbibition. DOG1 expression induced during seed imbibition cannot induce dormancy as a result of altered protein structures in after ripened seeds DOG1 interacting proteins Seed specific and clade A PP2C RDO5 AGH1/3 PDF1 In natural conditions seed dormancy shows a cycling pattern.  Romy Steenkamer Lectures 9 & 10 Seed germination chapter 4 Germination refers to the process starting with water uptake by the seed (imbibition) and ending with the emergence of the embryonic axis (usually as the radicle) through the surrounding structures (endosperm, perisperm, testa, pericarp). Two not mutually exclusive hypotheses for germination - Hypothesis 1: the growth potential of the embryo increases as the restriction of the seed coat is overcome by an increase in GA.  Romy Steenkamer - Hypothesis 2: the mechanical resistance of the endosperm reduces as a result of the absence of ABA Transcriptional changes during seed germination Testa rupture starts after imbibition of the seed. Genes are differently expressed in the embryo (cotyledons and radicle) on the hand and the endosperm (peripheral endosperm, chalazal endosperm and micropylar endosperm) on the other hand. Embryo and the endosperm have different fates. Transcripts are different both between endosperm and radicle as well as for the different germination phases. Genes that are downregulated during germination are upregulated during maturation and vice versa. It is possible that transcripts are present but their translation only occurs under favourable conditions. Translational changes during seed germination A transcribed mRNA is associated to many ribosomes, such a complex is called a polysome. Polysomes can be separated from the total mRNA pool on a sucrose gradient. Microarrays  Romy Steenkamer are used for RNA sequencing. In the graph on the right below blue depictures dry seed and reed imbibed seeds. The amount of polysomes is higher in the imbibed seeds as the level of translation is higher in these seeds. When the total amount of mRNA abundance correlates to the total amount of polysomal mRNA abundance, no translation regulation is considered to take place, as all produced mRNA’s are translated. 2 timepoints of translational regulation HTS = Hydration translational shift GTS = Germination translation shift Increased PO = number of upregulated genes Decreased PO = number of downregulated genes Translational regulation during germination is way higher than under stress conditions, which makes seeds a rather unique system.  Romy Steenkamer Priming: the prehydration of seeds followed by dehydration prior to planting, resulting in a more rapid and uniform germination and seedling emergence, improving crop establishment under stressful conditions as well. Germinative metabolism is increased after imbibition of primed seeds compared to non-primed seeds. The water potential is of the imbibition medium is decreased using glycol (PEG) or salts (osmotic priming) or the total amount of water provided to the seeds (hydropriming (matric priming), radicle emergence completion is prevented and seeds are stopped half-way. The major drawback of priming is that it results in a decreased seed longevity. Brassinosteroid-synthesis genes are differently expressed between accessions with different seed longevity’s. Brassinosteroids have a negative effect on longevity after priming. To prevent ion leakage or imbibitional damage in the hydration phase, dry membranes could be first warmed and melt by higher temperatures into the liquid-crystalline state before the entrance of water.  Romy Steenkamer Lectures 11 & 12 Social aspects of seed systems Seed is an essential element in the livelihoods of agricultural communities and important for income of market-oriented farmers. A seed system can be defined as all activities related to the production, storage, management, dissemination (distribution) and use of seed. A sustainable seed system ensures, seeds that are: o Of high-quality o From a wide range of varieties o Fully available in time o Affordable to farmers and other stakeholders Characteristics of a seed system The goal is multiplication as fast and as cheap as possible Multiplication systems of different crops differ in complication In many developing countries, seed is such essential element of the livelihood that it is a right: if you have no seed another farmer is obliged to give some seed to you Maintaining agrobiodiversity is essential, also for rituals Very large differences in references exist between men and women; - women (often caretakers of agrobiodiversity) prefer varieties that are easy to pound and to cook, do not taste too good (men will eat too much then) and are not easily digested - men (will be in charge of decisions once commercialization takes place) make market oriented decisions: colour or patterns of seed coats for examples. There are many different systems that help farmers to produce and/ or obtain seeds, in a given situation there might be parallel seed systems even for the same crop. Farmers mainly share seeds with their kin (tribe, family, religion, wealth status) Seeds ravel from rich to poor Formal seed systems: all activities are overseen by public and commercial actors, embedded in rules, regulations, laws and formal institutions. Seed certification is essential. Based on western models and do not work in developing countries since seed is a part of farmers’ cultures in these countries. Informal seed systems: no formal oversight, seed production is usually done by small- holder farmers, growing their own seed for neighbours, families and friends or even for markets. Often seed and ware cannot be distinguished (what is food and what is product). More flexible and diverse, accepting or creating more genetic diversity (using for example landraces). In general seed is from lower quality. Alternative of intermediated seed systems exist as well. High seed quality means proper physically, genetic, physiologically and healthy seed or seed that has a high ‘’fitness for use’’. Governance issues in seed systems are: - Inefficient seed production, resulting in impurities  Romy Steenkamer - Lack of quality control and certification resulting in poor physiological quality and seed health - Poor storage resulting in damage (insects), degeneration and loss of vigour - Access to cred to buy seed Summary -Many developing countries let room form parallel seed systems. - Agrobiodiversity plays a significant role in developing systems, especially for non- commercial smallholder farmers. - Seed is a social construct and a right, not just an input - Men and women have different roles in seed systems and are different in their variety preference. Common agronomic problems in Africa: Public pathogens Seed quality: - different in informal and from seed systems - high seed degeneration and low seed renewal Variety choice: traditional vs new cultivars Dormancy, especially in cases with multiple seasons per year Nutrient and water supply Continuum of seedborne pathogens Seeds flow from rich to poor. Men share seeds with other men. Women share seeds with other women. As more money is available in well-developed value chains, seed of better quality can be bought. Use of true potato is seed result in more seedlings than use of tubers as starting material. Reasons are the fact that the TPS yield more potato’s per plant (by breeding of seeds). On the other hand has positive selection (of tubers by farmers) a large insect resulting in better seedlings.  Romy Steenkamer Paradigms in seed supply systems An overlapping concept applied to rice farming in West Africa Lectures 13 Seed vigour Germination: the process happening in a seed after sowing, until the moment of radicle root protrusion Emergence: the seedling becoming visible above the soil Seed vigour: all the properties that determine the activity and performance of seed lots of acceptable germination in a wide range of environments. A vigorous seed lot is potentially able to perform well even under less optimal environmental conditions for the species or crop. A vigorous seed lot provides resilient seedlings that are able to deal with physical stresses (low moisture, oxygen, sanitation treatments) and biological stresses (competitions with weeds, pathogens, pests).  Romy Steenkamer High seed vigour is very important for the start of a crop and often also for the yield. Climate change demands more resilient cropping systems and thus also higher seed vigour. Influences on seed lot vigour Genetics Seed storage conditions (aging) Damage accumulated during production, treatments and storage o Seed production conditions o Seed harvesting methods o Seed treatments; sanitation, priming, sorting o Seed storage conditions Chlorophyll is degraded during late maturation; chlorophyll content can be used as a measure of maturation. Like a washing machine, seeds are delivered protected an unconnected at their new home. Mature seeds just shed from their mother plant are maximally protected. During priming part of the protection is removed thereby shelf life/ seed longevity is shortened. Seed vigour tests are used to provide information about the planting value in a wide range of environments and/ or the storage potential of seed lots. Energy (ATP) production requires a proton gradient over the inner membrane of the mitochondria. Unsaturated fatty acids in this inner membrane are prone to oxidation which can result in membrane damage. Damage in turn results in reduced aerobic respiration. As damage increases over time, the amount of aerobic respiration is a measure of seed aging. Less respiration indicates lower seed vigour. Respiration: o Tetrazolium: active mitochondria and active cell membranes will become red indicating aerobic respiration and intact cell membranes  Romy Steenkamer o Oxygen consumption: less respiration means lower oxygen consumption o Ethanol production: damaged mitochondria produce ethanol via fermentation Germination speed, decreases (obviously) for less vigorous seed Parameter Description Gmax Maximal activation level U7525 Uniformity parameter, time difference to reach 25% and 75% germination t50 The required time to reach 50% of the maximum germination AUC The area under the curve, as a measure of total germination Thermogradient Electroconductivity: membrane damage gives more leakage of amongst others electrolytes (sugars, amino acids, ions). Higher conductivity means less vigorous. Tolerance to controlled deterioration (CD) or Accelerated Aging (AA) Soil emergence and seed quality Higher vigorous plants are more tolerant to stresses. Less vigorous seedlings will be more infected. A plant is a holobiont: an assemblage of a host and the many other species living in or around it, which together form a discrete ecological unit. Plant growth promoting bacteria and disease resistance can be transmitted to the next generation through the seed microbiome. ‘Sterile’ seed production decreases germination, plant fresh weights and is accompanied by a shift in the microbiome composition. Seed sanitation used to focus almost only on the ‘bad guys’ in the seed microbiome (seed borne pathogens).  Romy Steenkamer Certain sanitation treatments may also remove ‘good guys’. Therefore present emphasis lays on removing exclusively pathogens by using natural biocides. The seed microbiome is part of seed vigour as it contributes to the performance of seed lots. Summary - Seed vigour is important for resilient cropping systems - Vigour is a trait with many characteristics - Vigour relates to field performance - Good performance for one condition is not necessarily good performance in another situation - Multiple methods exit to analyse seed vigour, mainly related to damage analysis: - Germination speed, - Respiration activity, - Cell membrane integrity - Seed production, treatments and storage all influence seed vigour Lecture 14 Seed drying and storage Eventually, seeds deteriorate during storage, which is unwanted by: - Seed companies and farmers because: - germination rate drops - Less vigorous seedlings - Total germination drops - Gene banks and breeders because: - Loss of seed quality - Loss of genetic variation The period of seed viability is greatly affected by: - genetics - quality at the time of collection - treatment between collection and storage - conditions of storage (temperature, relative humidity and oxygen) Higher storage temperatures and higher relative humidity increase deterioration of seeds. Graph shows at which equilibrium relative humidity and - temperature several processes can occur. The water activity ≈ % equilibrium RH/ 100 Equilibrium RH is influenced by temperature!  Romy Steenkamer Relative humidity of the storage conditions is more meaningful than seed moisture content: some parts of seeds (the oily parts) do not contain moisture, seeds with the same moisture content but different RH can differ greatly in being safe or unsafe for storage. Better to store seed in low temperature conditions (less optimal for bacteria and fungi, see graph). But RH can be quite high at low temperatures, solution: use closed containers. To maintain seed quality: Make seeds dry and keep them dry! Seed drying methods Natural methods: on the plant, in the wind Static drying: in the sun or shade Forced drying with air: heated air reduces RH Drying with moisture absorbers: only effective for small seed lots, for example: - silica gel, - salts, - drying beads, - rice Storing ultra-dry seeds is safe when stored in carbon dioxide or nitrogen instead of in oxygen. Deterioration types during harvest, drying and storage DNA damage Protein oxidation Lipid peroxidation Cell membrane damege Mitochondrial membrane damage All of these are oxidation types induced by reactive oxygen species (ROS). Reactive Oxygen species: arise from successive reduction reaction of oxygen. Dry seeds can survive better under anoxia: in oxygen depletion. Seeds can be stored under anoxia in vacuum packaging, in nitrogen gas or with the use of oxygen absorbers (iron powder). Seeds survive low oxygen levels better at low moisture content. At a low moisture level and temperature, sugars in the cytoplasm form a glass type of structure with a very low molecular mobility. With increasing moisture level or temperature,  Romy Steenkamer sugars will dissolve in the water and the cytoplasm becomes fluid with molecular mobility and enzymes become active again. Respiration only takes place when (mitochondrial) enzymes are active thus oxygen is only taken up when RH > 85% Seed lot shelf life tests - Humidity and increased temperatures stimulate seed deterioration → subject seeds to high moisture level and increased temperature to obtain a fast aging: o Accelerated aging tests: store seeds at high humidity and temperature and score survival after o Controlled deterioration tests: store seeds at a high humidity and temperature (but lower than for the AA tests), dry seeds back and retrieve germination data The correlation between the results of controlled deterioration tests and real seed storage experiments is quit poor because the physiology of seed stored at different moisture levels differs. Enzyme activity stops under low humidity, CD conditions thus do not predict seed longevity under commercial storage conditions correct. Increasing the oxygen concentration is an alternative dry experimental seed ageing method: Elevated partial pressure oxygen or EPPO store seed at high oxygen levels to mimic time. Summary - Seed quality is important for crop production and breeding and declines during storage. - Oxidation occurs in the presence of oxygen under dry conditions but pathogens thrive under humid conditions. - Deterioration rate can be limited by 1. Dry seeds and keep them dry (RH at 30%) 2. Store dry seed under anoxia 3. Hermetic package seeds 4. Store seeds cool 5. Water activity or eRH is a better indicator compared to seed moisture content - Seed physiology changes with water activity Ultra-dry seeds are more sensitive to membrane oxidation In the glassy cytoplasm state reduced oxygen levels can prolong shelf life In the fluid cytoplasm state, enzymes can become active At high humidity (>70%) seeds need oxygen for respiration. Estimating shelf life in AA tests, needs to take the water activity into account (including oxygen). - Oxygen interaction with RH: respiration takes place at RH>85% because then mitochondria become active. At lower moisture levels, sugars in the cytoplasm form a glassy structure  Romy Steenkamer with a very low molecular mobility and limit enzymes. - Seed quality loss during storage depends on the storage environment and starts as soon as seeds are dry. - The 3 main environmental factors are moisture, oxygen and temperature. - Drying the seeds is the first important step. - Anoxia storage is powerful but only if seeds are dry - Seed aging assays should mimic the RH of the storage condition - Seed physiology changes with the water activity: o Ultra-dry seeds are more sensitive to membrane (lipid) oxidation o In the glassy cytoplasm state reduced oxygen levels can prolong shelf life o In the fluid cytoplasm state enzymes can become active o At high humidity (.70%) seeds need oxygen for respiration o Estimating shelf life in AA, needs to take the water activity into account Lectures 15 Seed analysis and seed sorting The quality of seeds within one seed lot vary, due to: Other particles (stones, sticks) Damaged seeds Infected seeds Seeds with different shape Less mature seeds Seeds with different sizes Genetic variation Seed lots are populations that by nature differ in for example dormancy level, germination speed and longevity. Upgrading seeds lots demands the removal of lower quality seeds and strange objects. Traditional methods to do this used: sieves, gravity, indent cylinders, air, hand. Recent methods are based on fluid density or X-ray-, colour-, chlorophyll fluorescence image-, multispectral image analysis. Spectroscopic analyses terminology Absorbance: Logarithmic ratio of radiation falling upon a material, to the radiation transmitted through the material (also called optical density) Reflectivity: Fraction of incident radiation electromagnetic that is reflected at an interface Seed sorting based on fluid density, seeds on Transmittance: Fraction of incident electromagnetic radiation top of the fluid are often of bad quality. at a specified λ that passes through a sample Luminescence: Light emission by a substance not resulting from heat, but from a cold body, for examples chemical reactions, electrical energy Fluorescence: Light emission after light absorption, the emitted light has a lower energy (and thus a longer λ) than the absorbed radiation  Romy Steenkamer Chemical composition analysis uses near infrared radiation. Protein content and seed moisture content can be analyzed, non-viable seeds and insects can be removed. X-ray radiation detects cracks on seed coats. VideometerLab 19 LED’s are used to identify objects or pixels based on reflection spectra Pictures are taken at each λ Statistical software Example of the use of absorbance spectra analysis of seeds. Chlorophyll fluorescence analysis of seeds During maturation, chlorophyll is broken down. Higher fluorescence are an indication of immature seeds, which will result in less germination iXEED DataCollector: a tool that collects multiple types of radiation data (X-ray, visual, chlorophyll, hyperspectral) to analyse seeds. Terahertz analyses: can penetrate relatively deep in tissues and is used to discriminate certain varieties based on seed spectra. Raman spectroscopy analyses: sending a monochromatic light (of a single λ) on a sample and analyse the returning scattered light. Components such as fatty acids, proteins and lignin can give different spectra. Delayed luminescence or delayed fluorescence is the slowly emitted light from a sample in a few to a several dozen of seconds after stimulation by a light pulse. Summary - Seed analysis and sorting are distinct methods - Phenotyping (quality analysis) is part of both methods and is preferably based on individual seeds - Analysis is not always needed prior to sorting: gravity, size or liquid density based sorting - Chlorophyll fluorescence analysis can provide information about the maturity distribution  Romy Steenkamer of a seed lot - Analysis information can be used in seed sorting application: removal of less mature seed - Possibilities to analyse and sort seeds on quality are increasing: instrument costs are decreasing, computational data analyses is getting faster, single seed analyses will aid in recipe development. Present available techniques: Future - Visible light/ multispectral - 3D X-ray - Near infrared - Raman spectroscopy - X-ray - Terahertz - Chlorophyll fluorescence - Delayed luminescence Lecture 16 & 17 Management of plant genetic resources The ex situ approach and its relation with in situ and on farm management Protection of future food security: The potential amount of produced food is enough to feed the world (> 2000 kcal/ cap/ day). Food wastage ≈ 30% losses/ year Meat & dairy production ≈ 25% losses/ year Crop improvement ≈ 1.2% gain/ year - Cultivation - Breeding/ genebanks Genebanks are essential by giving the availability of sufficient genetic variation which is needed for the development of new cultivars and innovative research. Furthermore, genebanks aid in biodiversity conservation, while a worldwide decline takes place. The four pillars of food security Availability: food supply via production, distribution and exchange. Determined amongst others by land ownership, use, soil management, crop cultivation, breeding, harvesting. Access: affordability and allocation of food. Hunger and malnutrition are often not determined by food scarcity but by inaccessibility or food due to poverty.  Romy Steenkamer Utilization: the metabolism of food by individuals. Quantity and quality are determined by preparation, processing, cooking and nutritional value. Stability: ability to obtain food over time. Determined amongst others by seasonal factors, instability of markets, unemployment. Ex situ management: the conservation of genetic resources outside their natural habitat but inside storage organizations. Storage facilities differ between fields to the genebank of Svalbard at Spitzbergen. History class of genetic erosion 1890 – 1910 Value of landraces in relation to bred varieties was reported Warning about the loss of local landraces by replacement of uniform bred cultivars could lead to serious reduction in the genetic crop resource base. 1920 – 1930 First signs that genetic erosion is a worldwide occurring phenomenon. Development of first genebanks 1940 – 1950 CIMMYT (1943; wheat and maize) and IRRI (1959; rice) were established. Green revolution. 1960 – 1970 Plant genetic resources came on the political agenda: awareness about the importance its of collection and conservation. A global genebank network was established. Ex situ genebank management Acquisition Sources of accessions or seed samples, can be research organisations, companies botanical gardens other genebanks or expeditions.  Romy Steenkamer Prior to an expedition the national focal point (NFP) should be contacted to obtain prior informed consent (PIC) and negotiate mutually agreed terms (MAT). Local experts should be contacted for guidance in collecting. Furthermore, some basic information is needed: - Species appearance and distribution area - Number of populations that need to be sampled, depends on variation within and among populations. - Number of plants per population, from which should seed be collected, depends on breeding system and ploidy level. (See table for the Lawrence-Marshall-Davis approach: 60 for self and 30 for cross-fertilizing species) - Bulk seeds of one population or keep from individual plants separately. - Logistics: method of seed sampling cleaning, transport and vaccinations, medical kit, security. International regulations about exchanging plant genetic resources o Access and benefit sharing (ABS) arrangements: - Convention on Biological Diversity, focus on all users - International treaty on Plant Genetic Resources for Food and Agriculture, focus breeders and researchers. Regeneration Needed for accessions of too low seed quantity and/ or too low germination percentages. To avoid genetic shift (by natural selection) and genetic drift (by random allele losses), the regenerated seeds should have the same genetic content as the previous generation. For self-fertilizing species seeds from individual plants are sampled and stored separately. Individual plants are regenerated by selfing and progenies are kept separate → 100% maintenance of genetic variation over generations. Cross-fertilizing species should be regenerated by biparental crosses: plants between the progenies should be crossed and progenies should kept separated. (Example: collected 30 progenies, carry out chain crosses: female progeny 1 x female progeny 2, 2x3, 3x4, 29x30, 30x1) Genebanks mostly use bulk collection and -regeneration because: It is easier, quicker and cheaper than progeny separation; High frequency alleles (>0.3) can long be maintained; Variation will not decrease quickly over time if regeneration frequency is minimal; large populations should be used. Characterisation & evaluation To optimize the selection by users of accessions in a genebank information is needed: - Passport data: accession location - Characterization data: morphological traits - Evaluation data: resistance and genomic data  Romy Steenkamer Seed storage - Dry seeds - Determine initial germination % of an accession (cultivated: >80%, wild> 60%) - Long term storage: -20ºC, vacuum-sealed -Monitor germination percentage and seed quantity regularly Data management All collection management data are stored in a relation database, important for: - Control of the genebank system - Linking with international databases - Easy access for users Seed distribution & information - Accessions and related information are online available - Accession transfer only free for registered users On farm management The management of domesticated or cultivated species, in the surroundings where they have developed their distinctive properties. Landraces are maintained mostly by small farmers. Different approaches for on-farm management of agrobiodiversity: Conservation focussed: genetic resources conservation Development focussed: Farmers, local communities , livelihoods Community Biodiversity Management (CBM) combines both approaches: Farmers, local communities execute on farm management of crop genetic resources, while obtaining sustainable livelihoods and rural development is supported. - CBM is a community-based participatory methodology to strengthen community’s capacity through management of their knowledge based systems. - CBM aims to empower farming communities to identify, conserve, manage, add value and  Romy Steenkamer exchange crop diversity through community actions. - Key aim is to result in more delegation of authority to the community, develop ownership and support on-farm management of agrobiodiversity and sustainable livelihood options with minimum external inputs and risks. CBM tools: a lot of choices, amongst others: o Four cell Analysis : mapping local diversity in relation to households and area Effective for: - rapid assessment of diversity maintained - local monitoring - Red listing of cultivated crops at community level - Arising awareness to community about potential diversity loss - Developing local management plan o Social Seed Network Analysis: rapid assessment tool to map variety, seed and information flow at community level Effective for: - understanding who plays the key role in seed/varieties flow - identifying and recognising the yellow nodal farmers - supporting to design effective seed/ agrobiodiversity programmes. (First convince nodal farmers to use a new variety) CBM practices: again a lot of choices, amongst others: o Community seed bank: a community institution with short-term seed storage facilities. Effective for: - Local seed security social safety net - Access to seed and information of new, or local varieties - Effective local adaptation option to minimise climate risks - Linkage with ex situ gene banks o Participatory plant breeding: farmers, researchers and other stakeholders participation in various stages of crop improvement. Effective for: - Development of new varieties, suitable for local production Environment and meeting farmers needs - Conserving local diversity In situ management The conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species in their natural surroundings. History: Natural resource conservation exist for a long time but global biodiversity conservation is of recent origin. Regulation of the use of natural resources (resource ethics) became necessary to prevent selfish motives from taking more than could locally be sustained (which would endanger the  Romy Steenkamer long-term supply for the rest of the community). This social dilemma is often called the Tragedy of the commons. Resource ethics can be found in early writings from all over the world. Modern roots: Origin in situ management is found in the late 18e century (particularly UK and colonies). Conservation ethics is based on: - Human activities damage to the environment -Civic duty is needed to maintain the environment for future generations - Scientific, empirically based methods should be used to ensure this duty is carried out. First practically applied to forests in British India (1855) The term conservation came into widespread use in the late 19th century and referred to the management of timber, fish, game, pastures etc. mainly for economic reasons and the preservation of forest, wildlife, wilderness etc. 1950s Individual species are mostly conservation target 1972 United Nations adopt a programme to promote conservation of sites of unique or natural importance to the common heritage of mankind 1978 First international conference on research in Conservation biology 1992 Convention of Biodiversity (CBD), leading to the development of Biodiversity action plans in many countries Since 2000 Ecosystem approach (landscape scale conservation has taken over single species approach. Major components in the development of a conservation strategy Compile data on the target (species/ ecosystems) Priority setting: conservation goals for target Planning, design and setting-up conservation areas for target Management and monitoring of species/ areas Policy and legal support Final considerations - The use of much different strategies is possible - Plant genetic resources are the best conserved by using them - The conservation of (agro-) biodiversity depends to a large extent on the farming community and vice versa  Romy Steenkamer - Quantity and quality of conserved plant genetic resources depend on involved people - Plant genetic resource conservation will always be needed Lecture 18 Seed borne diseases and the general principles of seed testing Knowledge about the disease/ pathogen - Type of organism: viroid, virus, bacterium, fungus - Role of seed in infection: other sources of infection routes - Occurrence of the organism: geographical, incidence, specialisation - Symptom expression - Epidemiological features of the pathogen: biotrophic/ necrotrophic, monocyclic/ polycyclic, environmental influence Pathogens with a Q-status form the biggest threats. These bacteria and viruses are both difficult to detect as well as to eliminate. Seedborne bacteria Dissemination - Seed, -Wind driven splash dispersal, - Mechanically, -Insects Enter the plant via natural openings (hydathodes, stomata) or wounds Multiplication often favoured by high temperatures and high humidity Rarely soilborne Can survive and aggregate on leaves Symptoms of bacterial diseases: Seedborne Viruses Transmission - Seed, - mechanically (clipping, pruning, contact wounds), - via insects o Persistent: replication in vector o Semi-persistent: entering foregut o Non-persistent: only reaches distal end stylet Replication in plants or in insects Symptoms of viral diseases  Romy Steenkamer Seed-borne fungi Dissemination/ survival Penetration passively (wounds, natural openings) or actively (haustorium - Via wind or wind driven rain In moist conditions, spores are released causing a swelling and extrusion of fruiting bodies. Other fungi produce dry conidia, which are passively distributed by wind (>500m) or via droplets (5 -10 m) - Via insects - Via soil Fungi can survive in soil either free-living or in crop residues. Survival free in the soil is often short term survival (< 2months), although some fungi produce persistent resting bodies (e.g. sclerotia from Sclerotinia) which allows them to survive for longer periods (19 months). Survival in crop residues is shorter for crop residues in the soil than for crop residues on the soil. Necrotrophic pathogens can persist as long as the residues remain, biotrophic pathogens only as long as the crop is viable. Infection sources pathogens Basic seed/ planting material Soil/ soil debris Cultivation practices Weeds Irrigation water Air borne infections: rain, aerosols, wind Insects Type of organism Location on/ in seeds Viruses Embryo, sometimes testa/ endosperm Biotrophic fungi Embryo, some superficial Necrotrophic fungi Superficial, some in endosperm/ cotyledon Bacteria Superficial, some in endosperm/ cotyledon Pathogen longevity on seed is often within the time limits for commercial seed storage. The location of the pathogen (on or in seed) strongly influences its longevity. In seeds longevity is longer as pathogens are better protected. Locations can strongly influence the risk for primary infections, internal presence involves a higher risk because external inoculums are prone to adverse antibiotic effects. Storage conditions can influence longevity (dry, cool conditions are beneficial for pathogen survival). Effects of soil microflora on seed transmission of diseases A direct effect of soil type and environmental conditions influence the transfer from seed to seedling physical-chemical An indirect effect via micro-organisms - Percentage infected seedlings is higher in sterile soils than in unsterile soils - In dry soils with low bacterial activity → more fungal infections  Romy Steenkamer Seed transmission is higher in green houses and labs than in the field because in green houses and labs the conditions for the pathogens are more optimal. Seed health management before sowing Legislative measures and seed certification Quarantine measures - prevent entry - following outbreaks, prevent further spread & eradication of pathogen Seed certification programmes - establish tolerance levels - implement and control Good seed and plant practice (GSPP): aims to prevent tomato seed and plant lots from being infected by several pathogens (together called cmm) Isolation of the seed and seedling production location from the environment Prevention of infection by managing the 4 risk factors Constant monitoring during the growing seasons of seeds and young plants Check before delivery: all seed lots must be tested by independent people using seed test approved by GSPP The 4 risk factors - Water: management of production site must assure that the water is free of Cmm, if need disinfect. - People: to prevent contamination clothes are changed, hands are washed - Propagation material: has to be produced according to the GSPP standard - Materials: need to be disinfected before entering the production area Selection of seed production area’s Preferably in arid or semi-arid climates, as many fungi and bacteria are readily spread under cool moist conditions and by rain Greenhouse production to control insect- and wind-transmitted pathogens or to allow effective biological control  Romy Steenkamer General principles for seed testing Methods should be adequate for the target pathogen: sample size, number of subsamples, extraction techniques Standardized methods for various viral, bacterial and fungal diseases have been developed by ISTA and ISHI Results will be influenced by seed treatments Storage and packing can influence the seed health status A negative test result does not guarantee that the seed lot is pathogen-free, only that the seed sample tested was found to be negative Validated methods should be used that are: - Specific: distinguish target pathogen from all other organisms - Sensitive: able to detect pathogenic organisms in a low incidence in seed stocks. (Pathogens follow the Poisson distribution, thus the highest numbers have the lowest frequency) - Fast - Simple - Cost-effective - Reliable: robust and repeatable Inoculum thresholds: the level of infection on or in seed that will significantly affect disease development and result in economic loss. X = X0 * ert X = amount of disease X0 = initial inoculum r = infection rate t = time over which infection occurs Minimum tolerance level is a strategy based on reducing the initial inoculum X0 so that the inoculum threshold is not exceeded, even not under conditions favouring the infection rate (r). Crop Pathogen Minimum tolerance loss Lettuce Lettuce mosaic virus 1/ 30 000 Bean P. syringae pv. Phaseolicola 1/ 10 000 to 1/ 16 000 Cabbage Leptosphareia maculans 1/ 10 000 Cellery Septoria apiicola 1/ 7000 Onion Botrytis allii 1/ 100 Pea Ascochyta pis > 5/ 100 Field bean Didymella fabae >2/ 100 Lecture 19 Seed-borne diseases and an overview of on detection methods The correlation between field and seed lot symptoms is small making it necessary to do both. Direct inspection: Field inspections (of crops), looking for visual symptoms/ fungal structures on the seed eye with the naked eye and by microscope.  Romy Steenkamer Detection in seed Extracts - Seed extraction, for bacteria and viruses o Soaking: only useful for superficial pathogens o Stomacher o Grinding - Growth of pathogen o Agar testing o Blotter testing o Indicator plants Agar testing is not suitable for obligate (often biotrophic) fungi. Non-selective aga media: potato dextrose- or malt agar. Used to test which pathogens are present. Selective agar media: selective nutrients, physiological tolerances (salts, pH), selective antibiotics. Used to test if a certain pathogen is present. Blotter test is performed on viable seeds or on seeds with inhibited germination (by herbicides or freezing and thawing). From most sensitive to less sensitive - Detection/ identification o Symptoms/ macrostructures o Microscopical o Serological: ELISA/ Immunofluorescence cell-staining o DNA/ RNA amplification methods: TaqMan assay, LAMP assay, Next generation Sequencing New detection methods TaqMan assay: real time monitoring of PCR amplification - DNA extraction of seed extract - Specific forward and reverse primers for to be detected pathogen - If primer matches to the probe, polymerization releases the fluorescence from the TaqMan probe. - Moment of fluorescence signal is indicative for the amount of pathogen present - Can all be done in a closed system, no risk of contamination.  Romy Steenkamer LAMP-assay: Loop mediated isothermal amplification - Complicated, 4 different probes - One temperature, very fast - Can be done on site with small on-battery machines - Less prone to inhibitors, minimum extraction requirements are needed - Amplification can be done with both RNA and DNA templates (plant viruses are RNA viruses!) - But ~10-fold less sensitive compared with TaqMan real-time PCR - Primer sets can be combined and used for universal first and second line screening Next generation sequencing - Very fast de-novo amplification of terabytes - Can be used in a metagenomic approach: sequence all DNA in a seed extract and compare short contigs with those present in the databank (Not necessary to know beforehand what you want to detect). Seed health management in the field Culture measures and sanitation practices Crop rotations Sowing date: crop may outgrow pathogen at higher temperature Spacing of crops: use of disease-free areas (greenhouses) for production of basic seed Appropriate fertilisation Harvest time: just before maturity of seed (some pathogens only infect mature seeds) Roguing/ removing of diseased plants Control of weed and crop plant debris: could be survival places for pathogens Appropriate irrigation practices: furrow is better than overhead (results in aerosols) Disinfection of equipment and machineries Avoid wounding of plant material Use of pesticides to control diseases and pests Use of wind breaks Use of resistant cultivars for ‘’quality diseases’’ Use of tolerant cultivars (less desirable for quarantine pathogens as no symptoms will be visible in tolerant plant, so it is not sure whether the pathogen is present or not) Furrow irrigation Overhead irrigation  Romy Steenkamer Routes to internal seed infections Pollinating insects and dust created during combine harvesting can introduce pathogens as well. Seed health management during harvest and post-harvest Culture measures and sanitation practices Avoid cross-contamination during harvest, processing and storage Reduce infection levels in diseased seed lots by sorting and seed treatments Optimise storage conditions to suppress the progress of seed infections Upgrading seed lots Action Aim Risk Standard cleaning by Removal debris, sclerotia, spreading inoculum over separation hollow seeds equipment/ seeds Size grading Removal small potentially Loss of healthy seeds infected seeds spreading inoculum over equipment/ seeds Liquid cleaning Removal light seeds spreading inoculum over equipment/ seeds Electric eye cleaning Removal spotted seeds Masking invisible infections Physical treatment (e.g. Eradication of pathogens Reduction of vitality of seeds heat) Chemical treatment Suppression of pathogens Unwanted microflora shifts Inoculum could be eradicated and reduced chemically, physically and by biological means. Treating seeds with active ingredients as an alternative for spraying pesticides reduces the contact of the active ingredients with the environment and therefore has less impact on non- target organisms and drift.  Romy Steenkamer 2 types of ‘modern ‘fungicides’ Non-systematic (captan, thiram) - Limited penetration in seed - Not mobile within tissues of (germinating) seeds - Can protect against invasion of soil borne diseases - Eradicate pathogens superficially present on seed - Broad range of action - Low risk for resistance development (multisite inhibitor, cell poison) Systemic (carboxin, metalaxyl) - Prevent disease development away from application site - Eradicate deep seated seed infections - Mobile: translocated in sprouting seeds - Narrow range of action - High risk for resistance development (single site activity) Treatment of seeds Loading: correct ratio chemical to seed Distribution: uniform division between seeds Retention: strong adherence to seeds Hazard: no risk for operator Contamination: no pollution Physical methods to eliminate pathogens from seeds Hot water treatment Aerated steam Dry heat Radiation Electron treatment Pulsed-light Low-pressure-plasma Warm water treatment: relative simple and cheap method. Efficacy depends on temperature regime: quick raise should be followed by quick fall. Most effective against superficial organisms. Variations to improve efficacy: pre-soaking in cold water, addition of acidified cupric acetate or acidified zinc sulphate. Drawbacks: - Frequently incomplete eradication of bacterial and fungal pathogens - Ineffective against internal infection in larger seeds - Risk for seed damage - Requirement for redrying of treated seeds - Only small amounts of seeds can be treated at one time Hot steam treatment: the Thermoseed. Steam is applied to rotating seeds and after some time, seeds are dropped down and cooled with an air flow works very well, comparable to chemicals for cereals).  Romy Steenkamer Electron treatment: seeds are bombarbed with electrons, long durations of high voltages will harm the embryo. Higher voltages give, however, the best results. Biocontrol agents - Often less effective than chemical agents - Often more variation - Often short term protection But, as more and more chemicals are being banned, more and more intresting. Summary Preventive measures are prefered - Use of pathogen free basis seed - Grow in (semi) arid or contained area’s - Use of hygienic protocols strictly Use of curative seed treatments if appropriate - Upgrade seed qualtiy by sorting procedures - Treat seeds with suitable fungicides - Use physical treatments to control bacterial diseases