Têtè S Barigah

Hervé Cochard

Affliations Tete

S Barigah

Herve Cochard

Xylem Embolism Meter (Xyl'EM)

Keywords:

Tags: per cent

Background

Transpiration at leaf surface pulls water up from the soil into roots and through the xylem conduits of plants. Water in these conduits is under tension which increases as soil moisture content decreases and/or as transpiration rate increases. Water under tension can lead to xylem dysfunction by the breaking of water continuity in the conduit lumen. Embolism results from cavitation phenomenon, a process by which xylem conduits become air-filled and lose their functionality in sap conduction. Embolism rises in plants as a consequence of either freeze-thaw cycles during winter [START_REF] Sperry | Xylem embolism in response to freeze-thaw cycles and water stress in ring-porous, diffuseporous, and conifer species[END_REF]or air-seeding induced by water stress (Sperry et al. 1988a). Embolism can cause a substantial reduction in xylem transport and thus exerts a debilitating influence on plant water status by decreasing its hydraulic conductance. Besides, embolism is one of the most important threats to vascular plants, causing catastrophic xylem dysfunction when a threshold of water stress is passed (Tyree and Sperry, 1988). So far, embolism is commonplace in plants [START_REF] Milburn | Cavitation and embolism in xylem conduits[END_REF] and native steady state embolism and the related percent loss conductivity (PLC) are assessed by inserting excised stem segments in conductivity apparatus [START_REF] Cochard | Vulnerability of several conifers to air embolism[END_REF][START_REF] Magnani | Interpretation of seasonal changes of xylem embolism and plant hydraulic resistance in Fagus sylvatica[END_REF][START_REF] Sperry | Spring filling of xylem vessels in wild grapevine[END_REF].

The reference 'hydraulic' method for quantifying xylem embolism relied on a conductivity apparatus devised by (Sperry et al. 1988a). This method consists in estimating the hydraulic conductance of a stem segment before and after successive perfusions under pressure with degassed distilled water. The perfusion evacuates or dissolves air contained in the embolized xylem vessels. The initial to final conductance ratio gives a quantitative value of embolism level i.e. its steady state embolism and the related percent loss conductance (PLC). This technique is now wide-spread but remains laborious and delicate as it requires the use of a precision balance, and is unusable in the field.

The following protocol explains how to achieve a stem-segment (root and/or petiole as well) hydraulic conductance per unit pressure gradient measurement with the Xyl'Em.

Materials/Equipment

• Xyl'EM apparatus

Units, terms, definitions

The hydraulic conductance (k h , kg s -1 MPa -1 ) is known as the inverse of the hydraulic resistance (k = 1/R) and is the water flow rate (F, kg s -1 ) across a plant component divided by the pressure (P, MPa -1 ) causing the flow. k init and k max stand for initial and maximum conductances.

Native steady state embolism is expressed as

K h = F/∆P/l (1)
Percentage Loss of Conductivity (PLC, %) is calculated from

PLC = 100 (k max -k init ) / (k max ) (2)
NB. Initial hydraulic conductivity (k init , kg s -1 MPa -1 m), should be determined by measuring the solution flow rate (from the water basket) perfused through the stem segment of l meter long at a pressure drop (∆P, MPa) of about 6 kPa.

Procedure Hook up the Xyl'EM for measuring hydraulic conductance in stem segment

The Xyl'EM requires to be hooked up on the one hand to a computer for running an implemented program to output the conductance data, on the other hand to a set of plastic tubes, a sample manifold and an in-line 0.22 µm-mesh membrane filter holder. To proceed:

1. Lay down the Xyl'EM on a bench between a computer and a basin of tap water 2. Connect the Xyl'EM to the computer via the RS232 wire cable (RS, Xyl'EM front panel) 3. Plug in both the computer and the Xyl'EM to power up them 4. Connect the temperature probe to the Xyl'EM and keep the sensor head emerged in the tap water contained in the water basin 5. Connect the in-line membrane filter holder to the inlet/outlet F connexion ports of the Xyl'EM (see the front panel) 6. Connect the feeding tank to the inlet water port (to fill the inner tank) via a 3-way stopcock using a Luer tube or a Tygon tube (a home made connexion is needed). Always, fill by gravity the inner tank with de-ionized, de-gassed and filtered (0.2 µm) 100 mM KCl l -1 and 10 mM CaCl 2 l -1 solution 7. Connect the compressed air provider to the Xyl'Em's air inlet 8. Connect the water basket to a 3-way stopcock 9. Connect one void of this stopcock to the W.B. (Water Basket) port with a Luer tube 10. Connect the last void of this stopcock to a Luer tube and keep its free ending under the tap water inside the basin 11. Connect the sample manifold to the outlet port with a Luer tube 12. Switch on both the Xyl'EM and the computer 13. Refer to the instruction manual to fulfil the inner tank, the connexion tubings as well as the sample manifold mainly if you are running this operation for the first time 14. Make sure to remove all air bubbles from the water circuitry 15. Insert the samples in the sample manifold 16. The set-up is completed and ready for sample conductance measurements.

Preparing the plant material

Plant material (stem, petiole, mature portion of roots) requires a great care from harvesting to inserting in the sample manifold. The segments must be collected and prepared in such way so as to minimize the introduction of additional air bubbles into the xylem (see (Sperry et al. 1988a) for more details). When possible, feel free to spray water over the foliage of the target shoot before cutting or remove the leaves from the shoot prior to pruning.

1. Cut a shoot as long as possible from the mother plant 2. Wrap it in a wet black plastic bag or keep the cut end inside tap water 3. At least a piece of 0.15 m long should be removed under water from the cut end of each shoot and discarded. When focusing on petioles, they should be cut under water.

Software set up

1. Turn on the computer connected to the Xyl'EM and run the program that power up the Xyl'EM 2. Select the "set up" option to access the configuration options 3. Customize this page according to the type of flow-meter in duty; provide the calibration coefficient for the flow-meter as well as for the temperature and pressure probes. Make sure to select the right flow-meter (Int/Ext) if your Xyl'Em is released with these options 4. Select the units of the probes 5. Tick the automatic or manual option accordingly to your requirement 6. Fill in options related to the scrutinising time, the number of samples and number of 'flushes' 7. Valid your selections 8. Before leaving this page, tick the "LP" option for dedicating the device for Percent Loss Conductance (PLC) determination. "HP" option must be select for using the device as a High Pressure Flow Meter (HPFM) 9. Switch to the "Measure" window 10. If necessary, one can change here the default scrutinising-time interval, the sample and flush numbers and the options regarding the flow rate, temperature, low and highpressure.

Measurement processing

Set up the zero flow

This consists in closing the water basket outlet and opening the manifold to vent 1. Turn the first 3-way valve to "Flow", the second 3-way valve to "0" and the third one to "LP" 2. Click on the offset L-Press button on the computer screen to record the zero low pressure-flow offset 3. Close the manifold vent

Measurement of k init

1. Turn the 3-way valve (manifold) so that the target sample could be perfused with the solution from the water basket 2. Turn the 3-way valve hooked to the water basket to let its content feeds exclusively the target sample 3. Click on the GO button on the computer screen 4. Acquisition of initial conductance (k init ) starts and its values display automatically in the values window when each scrutinising time is over 5. When the k init reaches a steady state value, the coloured column turns from red or orange to green 6. Clicking on the OK button on the computer screen leads to stop k init data acquisition.

In the meantime, k init mean and standard deviation values are calculated and displayed in the 'stat' window. Only the k init -mean value is displayed in the "kinit" column in front of the sample 1 line. 7. Turn the 3-way valve that feeds the first target sample to prevent perfusing inward it 8. Select the next sample as target by allowing the solution from the water basket to flow exclusively through this new target sample 9. Initiate a loop by activating steps 4 to 10 to complete the k init measurement of this new target sample (sample 2) 10. Go for the same procedures to record k init value for each of the samples inserted into the manifold Flushing the samples measured for k max measurement 1. Close the water basket outlet 2. Turn all the 3-way valves of the samples in the sample manifold to allow water flow through all the samples simultaneously. Keep the vent outlet locked 3. Turn the second 3-way valve on the Xyl'EM front panel to "air" to allow compressed air into the inner tank up to 0.2 MPa maximum. Turn this 3-way valve to "0" 4. Turn the third 3-way valve on the Xyl'EM front panel to "HP" for few seconds to perfuse all the samples with high-pressured solution up to 0.2 MPa maximum 5. Wait for few minutes to allow high pressure relaxation within all the samples. Then, close water entry for all the samples (by turning their 3-way valve) except for the firsttarget sample

Measurement of k h

1. Open the water basket outlet to allow exclusively solution flow towards the samples 2. Redo operations from steps 4 to 13 3. Go anew for flushing operations (steps 15-19)

Measurement of k max

1. Repeat operations related to steps 22 and 23 until k h does no longer increase.

2. Turn all the 3-way valves on the Xyl'EM front panel to "0" 3. Save the data to an Excel file 4. Remove the samples from the sample manifold and proceed to measurements of their length and the diameter of the proximal end.

Other resources

Notes and troubleshooting tips

• If the plant material is too thin, you can put Teflon tape around it to fit the insert tubing (sample manifold) • During the first flush, take the sample manifold out of the water to check for leaks around the insertion points.

Links to resources and suppliers

Flow measurement:

• Principle: Thermal mass flow measurement • Measuring range: 0.1-5 g/h, 0.2-10 g/h, 0.4-20g/h, 1-50 g/h, 2-100 g/h H2O (to be specified on the order).

• Accuracy: + / -1% full scale • Range span: 1 to 50

Low pressure generation and measurement:

• Principle: Water column • Measuring range: 1 .. 7 kPa typ, (10 kPa max)

• Accuracy: +/-0.2 kPa High pressure generation and measurement:

• • Analog to digital conversion (15 bits resolution) and RS232 interface for data transfer to a computer.

• XYL'EM software for DOS and windows environments. This software collects the data, calculates the corrections and determines embolism factor.

Literature references

Fig 1 :Fig 2 :Fig 3 :Fig 4 :Fig 5 :Fig 6 :Fig 7 :Fig 8 :Fig 9 :Fig 10 :Fig 11 :Fig 12 :

 123456789101112 Fig 1: Front panel of the Xyl'Em

••

  Principle: Pressurised vessel • Measuring range: up to 3 bar typ, 7 bar max. • Accuracy: +/-1 % full scale Temperature • Sensor: Pt100 probe • Measuring range: 0° C to 50° C • Accuracy: +/-0Vessel capacity: 1 litre • Max vessel pressure: 3 bars, (7 bars possible if filter is removed). Low pressure: • Vessel capacity: 100 ml • Max vessel pressure: 1 meter water high • Water filtration: 0.2 µm. Mechanical characteristics • Suitcase: IP65 • Dimension: 461x347x206 mm • Weight (empty vessel): 10 kg Electrical characteristics Power supply: 12 Vdc • Consumption: 900 mA • Electrical connection: Din 3 pins Computer connection (option):

Species on which this protocol has been used


Philippe C Nicot
email: philippe.nicot@avignon.inra.fr

Marc Bardin

Biological and integrated protection in the Mediterranean greenhouse: is disease management the weak link?

Keywords: biological control, plant diseases, commercial products

While biocontrol is commonly used for the protection of greenhouse crops against arthropods, continued reliance on chemical control against diseases is often considered as a hindrance to integrated protection. This paper presents a review of currently available biocontrol products and other alternatives to pesticides for use against plant diseases in the Mediterranean greenhouse. At least 12 biological active substances (micro-organism or plant extract) are commercially available for the control of soilborne pathogens and 14 against foliar diseases, in one country or more in the Mediterranean Basin. Future prospects and challenges are presented.

Introduction

Greenhouse production systems are often cited among the most successful and the most advanced for the implementation of integrated protection schemes. Indeed, tools for biocontrol of pests in the greenhouse have long been available [START_REF] Van Lenteren | A greenhouse without pesticides: fact or fantasy?[END_REF](van Lenteren, , 2012) ) and natural enemies are successfully used against major pests in an ever increasing proportion of greenhouse acreage [START_REF] Pilkington | Protected Biological Control" -Biological pest management in the greenhouse industry[END_REF]. In contrast, development of biological control methods against plant pathogens is a more recent endeavour [START_REF] Van Lenteren | A greenhouse without pesticides: fact or fantasy?[END_REF] and continued reliance on chemical control against diseases is considered to be a hindrance to integrated protection, as many products have harmful effects on natural enemies. Is disease management really the weak link of IPM in the Mediterranean greenhouse?

This paper presents a review of currently available biocontrol methods for use against plant diseases in the Mediterranean greenhouse and proposes perspectives on short term evolution of the situation.

Major diseases of Mediterranean greenhouse crops

The main diseases present on greenhouse crops were comprehensively reviewed in the landmark book edited by Albajes et al (1999). While new pests have emerged in the Mediterranean greenhouses since the turn of the millennium, often associated with emerging plant viruses they can transmit, the key bacterial and fungal pathogens have overall remained the same. Much of the use of fungicides in the greenhouse targets either powdery mildews or airborne diseases fostered by excessive humidity such as Botrytis diseases, downy mildews (and late blight of tomato) and various leaf moulds, scabs and blights or stem rots. More recently, the effective phasing out of methyl bromide in the European Union (EU) and other countries around the Mediterranean has marked an increase in the importance paid to soilborne plant pathogenic fungi and nematodes.

Biological and integrated protection against diseases of greenhouse crops

Although research on biological control of diseases has been a very dynamic field, with an estimate of over 12 000 papers published in international journals between 1973 and 2008, only a trickle of commercial products have reached the market [START_REF] Fravel | Commercialization and implementation of biocontrol[END_REF][START_REF] Nicot | Classical and augmentative biological control against diseases and pests: critical status analysis and review of factors influencing their success[END_REF]. In 2010, only 14 microbial biocontrol agents were included in the EU list of active substances (Annex I of Directive 91/414) against plant pathogens [START_REF] Nicot | Review of factors influencing the success or failure of biocontrol: technical, industrial and socioeconomic perspectives[END_REF]. Various analyses have provided clues to explain this situation, including technical, regulatory and commercial difficulties encountered in the production, registration and use of biopesticides (Kabaluk et al., 2010;[START_REF] Nicot | Classical and augmentative biological control against diseases and pests: critical status analysis and review of factors influencing their success[END_REF][START_REF] Nicot | Review of factors influencing the success or failure of biocontrol: technical, industrial and socioeconomic perspectives[END_REF][START_REF] Ravensberg | Is the climate really changing in favour of biopesticides?[END_REF].

To identify currently available commercial products in countries of the Mediterranean Basin, several sources of online information were accessed in addition to simple Google searches with appropriate key words. Key, publicly accessible resources used in this study included the "EU Pesticide Database" (http://ec.europa.eu/sanco_pesticides/) and national databases on registered plant protection products in several countries of the European and Mediterranean Plant Protection Organization (www.eppo.int), the "Bio-Pesticides Data Base" of the University of Hertfordshire (www.herts.ac.uk) and the "Directory of Biopesticides in Organization for Economic Cooperation and Development Countries" developed by Agriculture and Agri-Food Canada (www.agr.gc.ca). Collected data were cross-checked among several databases and, whenever possible, validated directly with representatives of the companies.

Currently available commercial biocontrol products for the Mediterranean greenhouse

At least 12 microbial preparations, six of which are based on species of Trichoderma, are commercially available for the control of soilborne pathogens of greenhouse crops in one country or more of the Mediterranean region (Table 1). Their application range encompasses a variety of fruit, vegetable and ornamental crops. Their recommended use is most often as soil drench, but several products may be mixed into greenhouse substrates, used to dip transplants or cuttings or as seed dressing. This represents a substantial increase over the situation observed only a few years ago. Several products initially registered by companies based in northern latitudes are now distributed in the Mediterranean region.

Another striking improvement is that many products are now available for biocontrol of key airborne diseases (such as Botrytis and the powdery mildews) of a variety of greenhouse crops (Table 1). Particularly interesting among them, are several micro-organisms (such as Bacillus sp.) with a wide range of action, covering a variety of pathogens on many different crops.

However, the availability of biocontrol products against diseases is not evenly distributed within the Mediterranean region. Greenhouse growers in Italy, Spain and Turkey have the widest choice (with at least 6 to 9 products targeting soilborne and airborne pathogens), while there seems to be no product on the market in several countries (Table 1).

Other alternatives to chemical control of diseases in the greenhouse

Besides biocontrol, a variety of cultural practices may be used by growers to reduce the impact of diseases in the greenhouse (see excellent reviews in Albajes et al., 1999;van Lenteren, 2000, for example). Over the years the arsenal of such methods has widened, including the availability of less susceptible varieties, avoidance of excess humidity and the use of grafted transplants with resistance to soilborne pathogens. Increasingly, more systemic approaches are considered, including for example the impact of fertilization or water stress on plant susceptibility to diseases (and pests) and on the efficacy of biocontrol agents.

Future prospects and challenges

Based on the recent evolution we have observed, it appears likely that further extension of the geographical range of registration will be sought for some of the existing products, as well as an increase in the numbers of crops and possibly diseases targeted by those products. More products, currently under development or registered for the protection of vegetable, fruit or ornamental crops in other parts of the world may also be suitable for use in the Mediterranean region. Among an example of 15 such products (Table 2), it is striking that most are directed at the control of foliar pathogens (including key fungi such as powdery and downy mildews) and several have a wide range of use that includes soilborne diseases. There may be others on the way as well, as the EU Pesticide Database presents several new microbial strains with a "pending" status for inclusion in Annex 1.

Intense research and tremendous progress in the comprehension of the interactions between the plant, its environment, the pathogens and the biocontrol agents show promise for the selection of better microorganisms or plant defence enhancing compounds and for the design of more efficient formulations and tactics of application.

Finally, biocontrol is generally considered as unlikely to result in the selection of resistant pathogens. However, past experience with microbial biocontrol against arthropods suggests that caution should be applied lest durability would become an issue when biocontrol is increasingly used against certain plant pathogens. Other future challenges may result from the increasing cost of energy (with consequences for example on humidity reduction measures in the greenhouse), shortage of water in certain regions and various effects of climate change.

Conclusions

The recent evolution in the availability of biocontrol products against diseases of greenhouse crops in the Mediterranean greenhouse is highly positive. Clearly, the trickle has now become a stream and future evolution could be even faster as several leading plant protection

Table 1 :

 1 Examples of commercially available biocontrol products against diseases of Mediterranean greenhouse crops

				Current usage
	Active substance	Commer-cial name	Company	Crops	Mode of appli-cation	Target diseases or pathogens	Country of registration*
				several
	Ampelomyces quisqualis	AQ10	Intrachem www.intrachem.com	fruit, vegetable and ornamental	foliar spray	powdery mildew	BG, CY, FR, GR, IT, SI, ES
				crops
	Bacillus amylolique-faciens D747	Amylo-X	Intrachem Italia www.intrachem.it	several fruit crops and vegetable	foliar spray	various foliar fungi	IT
	Bacillus megaterium	Bio Arc	Organic for Bio Technology	several fruit & vegetable crops	foliar spray	various foliar fungi	EG
	Bacillus subtilis	Baubak	Biontek Inc.	cucumber	foliar spray	downy mildew	EG
				several
	Bacillus subtilis GB03	Companion	Growth Products www.growthproducts.com	fruit, vegetable and ornamental	soil drench / spray foliar	various foliar fungi soilborne &	GR, IT, TR
				crops
	Bacillus subtillis QST 713	Serenade	Agraquest http://agraquest. com BASF Agro www.basf.com	several fruit and vegetable crops	foliar spray	various foliar fungi and bacteria	FR, IT, IL, MA, PT, TR
	Bacillus subtilis Y1336	Biobac	Atlantik Tarim http://www.atlantiktarim. com/en/Urunler.aspx	tomato	foliar spray	Botrytis cinerea	TR
				several fruit		various
	citric acid + mint oil	Fungastop	Agriprotec www.agriprotec.com.tn	and vegetable	foliar spray	foliar & soilborne	TN
				crops		diseases
				several
				fruit,	mix into
	Coniothyrium minitans	Contans	Prophyta www.prophyta.de/en	vegetable and	substrat e or soil	Sclerotinia diseases	FR,GR, IT, PT, ES
				ornamental	drench
				crops
	Fusarium oxysporum F047	Fusaclean	Arysta Lifescience	cucumber	mix into drench substrat e or soil	soilborne fungi	BG
	neem oil	Agrimor Stop	Agrimor IL Ltd www.agrimor.co.il	pepper	foliar spray	powdery mildew	IL
				several
	neem oil	Nimgard	Agron Agrochemicals www.agron.co.il	fruit, vegetable and ornamental	foliar spray	powdery mites mildew +	IL
				crops

companies are becoming players in this domain. There is a general consensus among various sources that the global market for biopesticides is set to increase steadily over the coming years. A 2.5-fold increase is expected in 6 years by and Markets (2012), for example, to reach $3.2 billions in 2017. The question about disease management being the weak link of IPM in the Mediterranean greenhouse could thus be soon part of the past.