Sustainable Agriculture

MAICh

Research interests of Sustainable Agriculture

NUTRIENT, WATER AND ENERGY BUDGETS OF OLIVE OIL PRODUCTION SYSTEMS

Responsible Scientific Investigators: E. Kabourakis (NAGREF, Heraklion),   P. Barberi (Univ. Santa Anna, Pisa, Italy),  Dr. I. Livieratos

Increasing demand for food quality and safety as well as sound management of biotic and abiotic resources towards a minimum environmental impact have enforced a 3-years collaboration with a leading Greek company (MINERVA) in the food market. Here, primary production critical points are targeted and experimentally tested: agrobiodiversity, nutrient, water and energy use, CO2 emissions. The initiative follows previous examples worldwide for food safety and quality (e.g. farm to fork, the Global Food Safety Initiative etc.) and tangible outcomes are expected to benefit significantly olive primary production in Greece and the Mediterranean. A Production Standard will be created to cover primary production, processing and packing of olive oil.

OLD AND ENDANGERED ORGANICALLY CULTIVATED GREENHOUSE TOMATOES. EFFECT OF THE HARVEST TIME ON THE FLAVOUR INGREDIENTS AND CONSUMER ACCEPTANCE

Responsible Scientific Investigator: A. Krumbein (Leibniz-Institute of Vegetable and Ornamental Crops, Grossbeeren, Germany), I. Livieratos (MAICh, Greece), Dr. U. Kopke (Univ. Bonn)

The study investigated the effect of harvest time of four old and endangered tomato cultivars (‘Ananas’, ‘Auriga’, Green Zebra and ‘Lukullus’), organically grown in a greenhouse, on flavour compounds and consumer acceptance (Fig. 1).

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Fig. 1. a) Old and endangered tomato cultivars investigated (from top to bottom: Auriga, Green Zebra, Ananas, Lukullus); b) consumers test of the four old and endangered cultivars.

Measurement of acid and reducing sugars content, aroma volatiles analysis, as well as repeated consumer test were applied. The flavour compound analysis showed that the levels of acid content were mostly not affected by the stage of ripeness, whereas sugars were increased at optimal stage. Consumer tests showed that differences were perceived in several visual and sensory aspects between harvest times and cultivars. Optimal harvest was mostly preferred, nevertheless early harvest was not rejected and in many cases regarded as equal to optimal. Sensory evaluation was poorly interpreted by the aroma volatile profile and only partly by the acid and reducing sugar contents, probably due to the special external characteristics of the tomatoes. Useful information was derived, regarding the extension of the rather short shelf life of these cultivars, essential for planning appropriate market strategies to guarantee their preservation.

Publications

Klein, D., Gkisakis, V., Krumbein, A., Livieratos, I. & Köpke, U. (2010). Old and endangered tomato cultivars under organic greenhouse production: Effect of harvest time on flavour profile and consumer acceptance. International Journal of Food Science and Technology 45, 2250-2257.

REPLICATION OF WHITEFLY-TRANSMITTED CRINIVIRUSES

Responsible Scientific Investigator: I. Livieratos (MAICh, Greece) & K. Kalantidis (Univ. Crete, Greece)

We have chosen whitefly-transmitted criniviruses as a model to study virus replication, analyze genomes and develop diagnostic tools. Crinivirus virus particles possess thread-like, flexuous, filamentous virions with a characteristic “rattlesnake” structure morphology due to the presence of a cluster of structural proteins at one tip of the virion (Fig. 1). Their bi-partite single stranded 5`-capped positive-sense RNA genomes (15.000-19.000nt) have an organization that is similar to closteroviruses but divided into two molecules. The complete nucleotide sequence of several crinivirus genomic RNAs have been reported over the last decade. Crinivirus infections cause alterations of the phloem parenchyma and companion cells and form of characteristic inclusion bodies.

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Fig. 1. a) Cucurbit yellow stunting disorder virus-infected cucumber plants in greenhouses in Western Crete; b) virus particle morphology; c) Bemisia tabaci whitefly vector of several criniviruses.

Tomato chlorosis virus (ToCV)-induced infections in greenhouse tomato cultivations are prominent in the Mediterranean area and the United States showing identical yellowing symptoms that cannot be easily distinguished from Tomato infectious chlorosis virus (TICV) infections. In a recent study, both genomic RNA components of a Greek isolate (Gr-535) of ToCV were sequenced and compared with Spanish and American isolates (Fig. 2). The prediction of putative structures of the 3’-terminus of ToCV RNA 1 showed the presence of four stem loops and a pseudo-knot. These structures get possibly recognized by the viral RNA dependent RNA polymerase during the initiation of ToCV RNA 1 negative strand synthesis. Diagnostic dot-blot hybridization and reverse transcription–polymerase chain reaction (RT-PCR) assays routinely and specifically detect the virus in 20ng of total RNA extracts from ToCV-infected plants. Dot-blot hybridization can also be performed for virus diagnosis using infected crude plant extracts.

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Fig. 2. Tomato chlorosis virus-infected tomato plants in a greenhouse in Western Crete A). ToCV bi-partite genome where the open reading frames are shown as boxes B). Dot-blot hybridization using a DIG-labelled probe corresponding to the minus strand of ToCV-p22 gene. Five-fold dilutions of total RNA (A & B) or plant (C & D) extracts from infected (A & D) and healthy (B & C) tomato plants were spotted onto nitrocellulose membranes. For total RNA extracts, 0.5ug (lane 1), 0.1ug (lane 2), 20ng (lane 3) were used. For plant sap, 10ul of 1/5, 1/25, 1/125 plant extract dilution (g/ml) in phosphate buffer was used. 20ng of a DNA clone of ToCV p22 was used as a positive control (top of the membrane). C). Secondary structure model for the 176 nt long 3’-UTR of the Greek ToCV isolate RNA 1. Stem–loop structures are denoted as hpI, hpII, hpIII and hpIV. Complementary sequences involved in the putative interactions are underlined and noted (A). Dotted lines indicate the pseudoknot interaction PK1 D).

Post-transcriptional gene silencing (PTGS) degrades RNA in a sequence specific manner and is utilised by plants as a natural defence mechanism against virus invaders. Using an Agrobacterium-mediated transient assay on Nicotiana benthamiana wildtype and 16c plants, we screened four Cucurbit yellow stunting disorder virus (CYSDV) RNA 1-encoded proteins (papain-like protease, p25, p5.2 and p22) to determine which one possess PTGS suppressor activity. Amongst these proteins, only CYSDV p25 was able to suppress (double- and single stranded) RNA-induced silencing of the green fluorescent protein (GFP) mRNA (Fig. 3). Restoration of GFP expression by CYSDV p25 in both of these experiments had no apparent effect on the accumulation of the small interfering RNAs.

Fig. 3. CYSDV p25 suppresses ssRNA-induced RNA silencing of GFP in Nicotiana benthamiana 16c plants A). N. benthamiana (16c line) leaves infiltrated with mixtures of Agrobacterium cultures harboring 35S-GFP either in combination with the pGreen empty vector (I) or with constructs expressing CymRSV p19 (II), ToCV p22 (III), CYSDV p25 (IV). UV light images were taken 8 or 20 days post infiltration (dpi) B). Northern blot analysis of GFP mRNA and siRNA extracted from patches agroinfiltrated as indicated above each lane at 14 dpi. Ethidium bromide staining of rRNA was used as loading control (upper panel). GFP mRNAs and siRNAs were hybridized with a random primed 32P DNA probe corresponding to the complete GFP ORF (middle and lower panel, respectively) C).

Publications

1. Kataya, A.R.A., Suliman, M.N.S., Kalantidis, K. & Livieratos, I.C. (2009). Cucurbit yellow stunting disorder virus p25 is a suppressor of post-transcriptional gene silencing. Virus Research 145, 48-53.

2. Kataya, A.R.A., Stavridou, E., Farhan, K. & I.C. Livieratos (2008). Nucleotide sequence analysis and detection of a Greek isolate of Tomato chlorosis virus. Plant Pathology 57, 819-824pp.

PEPINO MOSAIC VIRUS REPLICATION

Responsible Scientific Investigators: T. Osman (Univ. Fayoum, Egypt), R. Olsthoorn (Univ. Leiden, The Netherlands),T. Canto (CSIC, Spain), A. Makris (MAICh, Greece), V. Medina (Univ. Lleida, Spain), Livieratos (MAICh, Greece)

Pepino mosaic virus (PepMV), a member of the genus Potexvirus. PepMV was first reported from infected pepino (Solanum muricatum) crops in Peru and later detected in tomato crops in the Netherlands, France, Germany, Hungary, Italy, Poland, Spain, UK, USA and Canada. Symptoms induced by PepMV infection in tomato (Fig. 1) vary and are dependent on environmental conditions and virus strain. The symptomatology includes leaf mosaic, mottling, distortion such as “bubbling”, spiky or nettle-like heads, dwarfing, and fruits with a marbling effect or tiger-stripe symptoms.

Fig. 1. Tomato leaf (A) and fruit (B) symptoms in plants infected with PepMV

(www.agf.gov.bc.ca/cropprot/tomatoviruses)

We used PepMV-encoded proteins as baits to screen a tomato (Solanum lycopersicum) cDNA library for potential interactors in yeast (Fig. 2A and 2B). Potential interactors were selected and used to confirm the interaction in various assays in vitro and in planta (Fig. 3A and 3B).

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Fig. 2. A). Principle of the yeast two-hybrid system: reconstitution of a functional transcription factor via a protein-protein interaction. The DNA binding domain (DBD) of the transcription factor is expressed as a hybrid protein fused to protein X ("bait"); the activation domain (AD) is fused to protein Y ("prey"). Only if X and Y interact will the activation domain be in the proper position to activate transcription of the reporter gene (www.rzpd.de/services/two_hybrid/principle). B) Confirmation of positive interactions between PepMV p25 (A and B), PepMV CP and respective selected primary positive clones (C and D).   A and C: Gal-Raff/CM-His-Trp-Leu plates; B and D: control Glu/CM-His-Trp-Leu plates. The red arrow indicates EGY48, pGILDA/p25 or pGILDA/CP re-transformed with the respective positive clone prey plasmid DNA. The white arrow indicates EGY48, “empty” pGILDA re-transformed with the respective positive clones prey plasmid DNA.

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Fig. 3. A). Interaction and subcellular localisation of Hsc70s and PepMV CP in living N. benthamiana cells by Biomolecular Fluorescence Complementation (BiFC) assay. B) Double IGL of PepMV-infected phloem tissue using Hsp70/Hsc70 and PepMV CP antiserum.

On a different note, PepMV in vitro template-dependent system for the study of viral RNA synthesis has been established. These two systems are used to identify in vitro and verify in vivo essential elements and co-factors for the initiation of PepMV negative-strand RNAs.

Publications

1. Osman, T.A.M., Olsthoorn, R. & Livieratos, I.C. (2012). In vitro template-dependent synthesis of Pepino mosaic virus positive- and negative-strand RNA by its RNA-dependent RNA polymerase. Virus Research (in press).

2. Mathioudakis, M., Veiga, R., Ghita, M., Tsikou, D., Medina, V., Canto, T., Makris, A.M. & Livieratos, I.C. (2012). Pepino mosaic virus capsid protein interacts with a tomato heat shock protein cognate 70. Virus Research 163, 28-39.


GLYPHOSATE RESISTANCE OF HORSEWEED (CONYZA CANADENSIS) COLLECTED FROM CRETE

Responsible Scientific Investigator: C. Giannopolitis (BPI, Greece), I. Livieratos (MAICh, Greece)

Glyphosate (N-phosphonomethyl glycine) is a foliage active herbicide which controls a broad spectrum of annual and perennial weeds. Since its introduction in 1974, glyphosate became world’s dominate herbicide. The widespread use of glyphosate has increased selection pressure on many weed species and several of them, including horseweed (Conyza canadensis) have expressed resistance. Resistance to glyphosate in horseweed was first reported in the United States in 2000, while resistant biotypes have been reported from other states of the United States but also Brazil, China, Australia and Europe. Glyphosate inhibits the enzyme involved in this pathway: 5-enolpyruvyl shikimate- 3 phosphate synthase (EPSPS). EPSPS is the critical and essential enzyme that catalyzes the conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP. Inhibition of EPSPS reduces the biosynthesis of the aromatic amino acids (phenylalanine, tyrosine and tryptophan) and leads to plant death. Glyphosate initially causes accumulation of shikimate-3-phosphate, the substrate of EPSP synthase, which is then hydrolyzed in the plant to shikimate (Fig. 1).

Fig. 1. The shikimate pathway and mode of action of glyphosate

Detection of shikimate in plants can be used to determine whether a plant has been exposed to glyphosate and can also be used to determine whether plants are resistant. Conyza canadensis (horseweed) plants. Twenty-two biotypes of C. canadensis from a conventional orchard in Crete displayed varying degrees of reduced glyphosate susceptibility in standard whole plant assays (Fig. 2).

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Fig. 2. a) Horseweed in a conventional citrus orchard in Crete; b) individual conyza plants in the greenhouse (G0); c) conyza seed collection; d) production of different horseweed biotypes for testing in cotyledon stage; e) at the seedling stage; f) at the rosette stage.

A refined shikimate leaf disc assay was developed to precisely determine the resistance levels, permitting early detection of resistance evolution and integrated management of the weed. The EPSPS homologue genes (1 and 2) were sequenced for three different biotypes (one of reduced susceptibility from Crete, one resistant from mainland Greece and one resistant from the USA), and no amino acid substitution of Pro106 was found. Real-time qRT-PCR was used to study the expression profiles for EPSPS and the M10 and M11 ABC transporter genes, following glyphosate application. The expression levels of the EPSPS genes were not significantly altered following glyphosate application in any biotype, but both M10 and M11 were found to be highly upregulated in glyphosate-treated reduced susceptibility or resistant biotypes and not in a susceptible biotype. These results are in accordance with data recently reported by other researchers, supporting a role of the M10 and M11 ABC transporter genes in glyphosate resistance in C. canadensis, because of reduced translocation.

Publication

Nol, N., Livieratos, I.C. & Giannopolitis, C.N. (2012). Reduced susceptibility to glyphosate of Conyza canadensis plants from a conventional citrus orchard in Crete (Greece) and early detection of resistance with the shikimate leaf disc assay. Weeds Research 52, 233–241.


SEASONAL POPULATION TRENDS AND HOST PREFERENCE ON 8 COMMON HOST PLANTS OF CALOCORIS TRIVIALIS UNDER FIELD CONDITIONS

Responsible Scientific Investigator: A. Kalaitzaki (NAGREF, Chania, Greece)

Calocoris trivialis is a phytophagous species which is widespread in the olive groves but also in citrus orchards in Greece and in the rest of the Mediterranean region. It is a secondary entomological pest of olives and causes local and occasional damage of economic importance. The last 10 years, under specific conditions, it caused sporadic damage of economic importance in certain olive-growing regions of Chania prefecture. Nymphs and adults feed on buds and blossoms which results in the premature dropping of buds and of flowers respectively. Aim of a study was to evaluate the fluctuation of the population of the insect (seasonal appearance) by field experiments. Weekly samplings from olive groves and citrus orchards will take place in order to estimate the fluctuation of the population of C. trivialis from January until May. Host plant preference were studied (density of the population of the insect to the various plant hosts that are existing in the olive groves and in the citrus orchards). Weekly samplings took place from weeds, olive groves and citrus orchards. All samplings will be carried out between January and May. The economic injury estimation will be defined. It will also be studied the development time of immature stages of Calocoris trivialis at three temperatures (15, 20, 25oC) and its oviposition rate at 25oC on the host plant Sinapis alba with laboratory experiments.

Fig. 1. Adults of Calocoris trivialis.

http://www.agriwww.it/Agronomia/immagini/Malattie/C9.jpg

http://www.flickr.com/photos/13703321@N07/2801993267/

Publication

Amara, A. & Kalaitzaki, A. (submitted). Effect of temperature on development time of Calochoris trivialis. Journal of Pest Science.


RESPONSES OF CUCUMBER TO COMPOSTED PIG MANURE USED AS SOLE SUBSTRATE OR IN MIXTURES WITH PERLITE AT DIFFERENT RATIOS: IMPACT OF THE HEIGHT OF THE SUBSTRATE IN THE BAG

Responsible Scientific Investigators: D. Savvas (AUA, Greece), A. Stamatakis (MAICh, Greece), I. Livieratos (MAICh, Greece)

The cultivation of vegetables on substrates is an efficient alternative to soil sterilization, especially in view of the phase out of methyl bromide in compliance to the Montreal protocol. On the other hand, the public pressure to recycle organic by-products originating from agriculture and industry is increasing. In most countries, large amounts of agricultural organic waste originate from the livestock sector and release of large amounts of animal manure to the environment results in extensive pollution of water resources. As a result, livestock producers constantly face the challenge of managing manure and meeting environmental regulations. One of the viable and environment-friendly possibilities to dispose of livestock manure is to use it in agriculture. In addition to its use as an organic fertilizer in soil-grown crops after composting, livestock manure can be used also as a substrate for soilless cultivation of productive greenhouse crops.

During a 3 years project with a leading company in the food sector (CRETA FARM), we evaluated the use of Creta Fert pig manure in hydroponics. Cucumber plants grown on different ratios of composted pig manure and perlite and different bag heights (10, 20cm). In these experiments, sole compost placed in bags at a height of 10 cm gave significantly higher yield and average fruit weight than 20 cm height bags, and statistically surpassed sole perlite. Cucumber plants grown on compost gave a higher total yield or number of fruits compared with those grown on sole perlite (Fig. 1).

Fig. 1. Initial data (total fruit weight [A] and total number of fruits [B]) from an early harvesting of cucumber plants grown on different substrates.

Publication

Al Naddaf, O., Livieratos, I., Stamatakis, A., Tsirogiannis, I., Gizas, G. & Savvas, D. (2011). Hydraulic properties and agronomic performance of perlite are improved when mixed with composted pig manure. Scientia Horticulturae 129, 135-141.

 EXTENSION IN ORGANIC AGRICULTURE: CHANGING NEEDS IN TRAINING TOWARDS FACILITATION OF LEARNING.

Responsible Scientific Investigators: A.Koutsouris (AUA, Greece),  E. Kabourakis (NAGREF, Heraklion, Greece), I. Livieratos (MAICh, Greece)

 With the growing relevance of organic agriculture, input intensive conventional farming systems are replaced by knowledge intensive organic farming systems. Developing and performing appropriate interventions in organic agriculture is based on a complex learning progress. Instead of the linear extension models prevailing in conventional agriculture and providing universal solution and transferring knowledge from scientists to farmers, extension for organic agriculture should enhance farmers’ observation skills and their own judgments and decision making. Thus, not only the farming systems are underlying a change, but also the requirements for high quality extension work are changing and methods have to be adapted. Currently, extension services in Europe for organic farmers are provided by various governmental and private institutions. It’s ranging from combined extension for conventional and organic farming up to specialized in organic farming, from expert consultants to farmer self-help groups. Thus, performance of extension is varying considerably in quality and methods.

In order to know how extension for organic agriculture can be improved, we investigate the current status of it and to define satisfactory and unsatisfactory elements of it:

-          Defining the current status of applied participatory approaches

-          Who provides extension services to organic farmers and how do organic farmers access information in Germany and Crete

-          Which methods are used for extension in organic agriculture and what are the differences compared to conventional farming?

-          What are key elements for successful facilitation of learning and how can the training be improved?

Information on these topics are gathered through literature reviews and by conducting interviews with institutions representatives and experts involved in research. A comparison between the situation in Germany and in Crete is also carried out.

Last update: Oct 23, 2014

Sustainable Agriculture