WO2014001476A2 - Traitement thermique non destructif d'arbres pour arrêter la progression de maladies - Google Patents

Traitement thermique non destructif d'arbres pour arrêter la progression de maladies Download PDF

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Publication number
WO2014001476A2
WO2014001476A2 PCT/EP2013/063567 EP2013063567W WO2014001476A2 WO 2014001476 A2 WO2014001476 A2 WO 2014001476A2 EP 2013063567 W EP2013063567 W EP 2013063567W WO 2014001476 A2 WO2014001476 A2 WO 2014001476A2
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aesculi
bacteria
bacterial
tree
plant
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PCT/EP2013/063567
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WO2014001476A3 (fr
Inventor
Andreas Antonius Maria VAN LAMMEREN
Jeroen DE KEIJZER
Matteus Johannes KETELAAR
Alphonsus Joannes VAN KUIK
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Wageningen Universiteit
Stichting Dienst Landbouwkundig Onderzoek
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Priority claimed from GBGB1211638.0A external-priority patent/GB201211638D0/en
Priority claimed from GBGB1211710.7A external-priority patent/GB201211710D0/en
Application filed by Wageningen Universiteit, Stichting Dienst Landbouwkundig Onderzoek filed Critical Wageningen Universiteit
Priority to EP13732160.0A priority Critical patent/EP2866550A2/fr
Publication of WO2014001476A2 publication Critical patent/WO2014001476A2/fr
Publication of WO2014001476A3 publication Critical patent/WO2014001476A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants

Definitions

  • the invention relates to the field of phytopathogenic control; more particularly in woody species such as shrubs and trees.
  • aesculi is found as an epiphyte on leaves, flowers and fruits of the host tree [9].
  • the bacterium can adopt an endophytic, pathogenic lifestyle in the presence of small wounds, for example leaf scars or superficial injuries caused by human activities [10,1 1]. Further potential entry points for the bacterium have been shown to include lenticels
  • Wounded plant tissue can lead to desiccation and forms a potential entry point for pathogens. Therefore, it is of importance for plants to separate healthy from disrupted tissues.
  • wounds or infection sites in tree barks can be sealed off by de novo periderm (consisting of phellem, phellogen and phelloderm) formation, which is considered a non-specific defence response [12].
  • periderm consisting of phellem, phellogen and phelloderm
  • Formation of a new periderm layer near infection courts in Aesculus bark has been observed, but appeared ineffective in preventing (further) bacterial invasion [4,11].
  • cell wall reinforcing components are deposited by existing cells near wounds in many tree species [13-16].
  • lignin and suberin are found as widespread components of initial wound-repair and may function in preventing water loss and further tissue collapse [13].
  • lignin and suberin are found as widespread components of initial wound-repair and may function in preventing water loss and further tissue collapse [13].
  • For horse chestnut the timing and nature of such structural cell wall reinforcements in a wound boundary zone have not been described and its possible role in preventing the development of bleeding disease is unknown.
  • the present invention provides a method for the prevention or control of bacterial infection of tree species, comprising exposing a tree species or part thereof to a temperature not less than about 39°C for a period of time of 48 hours or more.
  • the method provides a practical approach to treatment of woody species in situ.
  • the invention provides a method for the prevention or control of bacterial infection of tree species, wherein the temperature is maintained substantially constant during the period of exposure. In preferred methods the temperature is maintained at 39°C for a period of 48 hours.
  • the tree species, part thereof or seed may be exposed to the temperature by conductive heat transfer from an heat source; and/or radiative heat transfer from an heat source; optionally there being two or more sources of heat.
  • the heat source may be lamps (e.g. infra-red lamps), hot air blowers, radiators, heated bars or coils and/or tubes filled with hot air, liquids or gels.
  • lamps e.g. infra-red lamps
  • the plant part or branch or trunk may be coated or painted with a radiation adsorptive layer which for aesthetic reasons of treating trees e.g. in parks and gardens is preferably removable, e.g. matt black paint which is preferably water soluble.
  • the tree species or part thereof treatable by the method may be exposed to the temperature for the period of time by (a) placing the tree species or part thereof or seed into a temperature controlled chamber; and/or (b) placing an heat source in direct contact with at least a portion of a woody part of the tree.
  • the source of heat may be a flexible linear heating element wound around the circumference of the woody part.
  • the flexible linear heating element is a fluid filled tube and heated fluid is pumped through the tube.
  • Temperature sensors may be used to control the supply of heated fluid to the heating elements to achieve substantially uniform temperatures for the desired length of time.
  • bacterial infections treatable by the method can include but are not limited to infections caused by bacterial species selected from the following list; Pseudomonas syringae pv. morsprunorumplum in plum (Prunus domestica) or cherry (Prunus avium), Pseudomonas syringae pv. savastanoi (e.g. Pseudomonas savastanoi) in olive (Olea europaea), Pseudomonas syringae pv. actinidiae in Kiwi (Actinidia deliciosa) or Golden Kiwi (A. chinensis), Xanthomonas citri pv.
  • citri i.e. Xanthomonas axonopoulos pv. citri
  • Citrus spp. or Citrus hybrids Xanthomonas arboricola pv. pruni in laurel cherry or bay cherry (Prunus laurocerasus), peach (Prunus persica) or almond (Prunus amygdalus), Erwinia pyrifoliae in Asian pear tree (Pyrus pyrifolia) or Pyrus communis or Erwinia amylovora in species of subfamily Maloideae (family Rosaceae).
  • the bacterial infection is caused by Pseudomonas spp., Xanthomonas spp. or Erwinia spp. and more particularly, is Pseudomonas sp. infection of Aesculus spp.
  • the bacterial infection is Pseudomonas syringae; preferably Pseudomonas syringae pv. aesculi infection of Aesculus hippocastanum.
  • the plant or plant part may exhibit Horse Chestnut Bleeding Disease.
  • Plants or plant parts obtainable by the method described may be free of viable bacterial pathogen.
  • Aesculus hippocastanum, a part thereof or seed obtainable by the method herein described are substantially free of viable Pseudomonas syringae pv. aesculi.
  • the present invention also provides an apparatus for heating a woody tree or branch comprising a fiexible heating element connectable to a power or heat source, characterised in that in a first position prior to application in use to a tree or branch the fiexible heating element is substantially linear, and in a second position in use the fiexible heating element is substantially in the form of a coil around and in intimate contact with a circumferential portion of the tree trunk or a branch.
  • Pseudomonas syringae pv. aesculi strains (listed in table 1) were grown in Lysogeny Broth (LB; [47]) at 28 °C and 180 rpm. E. coli strains (Table 1) were routinely cultured in LB at 37 °C and 225 rpm. Plasmid pMP4655 (obtained from the Department of Molecular Microbiology, Leiden University; Table 1) was introduced in E.coli DH5a and GM119 by electroporation. Antibiotics were added in the following concentrations unless stated otherwise: tetracycline, 40 ⁇ g/mL; carbenicillin, 100 ⁇ g/mL and kanamycin, 50 ⁇ g/mL. Strains were stored at -80 °C for long-term conservation.
  • Transformation of P. s. pv. aesculi by triparental conjugation The two E. coli donor strains harbouring pMP4655, helper strain cel40 containing pRK2013 and recipient P. s. pv. aesculi strains were grown until late log phase. Antibiotics present in the initial culture medium were first removed by collecting cells at 1,000 x g for 5 min and resuspending them in their original culture volume of LB. All cultures were pelleted again and resuspended in 3% of their original culture volume of LB.
  • Cb carbenicillin
  • Km Kanamycin
  • Tc Tetracycline
  • Tra conjugal transfer functions
  • Plant cultivation and inoculation For the analysis of the early defence response and bacterial invasion upon inoculation, experiments were conducted with one and two-year old horse chestnut (A. hippocastanum) seedlings. Plants were maintained at room temperature under natural light in containers with 5 L potting soil and watered weekly. Experiments were repeated three times. Eight plants in total were inoculated at various sites on the stem for sampling in time, two inoculations at opposite sides of the stem. At each sample moment (see 'Sampling, fixation and sectioning ' ') one set of opposite inoculation sites was removed. Plants were either mock-inoculated as a control (3 plants in total) or inoculated with P. s. pv. aesculi (5 plants in total).
  • a superficial wound was inflicted on a surface sterilized part of the stem by making a small (2-3mm), diagonal cut with a downward angle using a sterile razorblade. Care was taken not to cut deeper than the cambial zone.
  • These wounds were inoculated by pipetting 5 of sterile PBS or 5 PBS containing 10 cfu/mL P. s. pv. aesculi PD4818 into the wound fissure.
  • Infection experiments with GFP expressing bacteria were performed on three separate occasions in climate cells using 6 two-year old horse chestnut seedlings maintained at 21 °C, 70-90 % RH and a 16 h light, 8 h dark cycle. Plants were sampled after 4 and 6 weeks to examine in planta behaviour of bacteria, and monitored over a period of 8 weeks.
  • samples of stem were removed from the inoculated seedlings around the original inoculation site at 0, 1, 2, 3, 4, 6, 8 and 14 days after inoculation. For investigation of later stages, samples were taken at 2, 3, 4, 10 and 18 weeks after inoculation.
  • the sampled pieces of stem were fixed in 4% w/v paraformaldehyde, 0.05% v/v glutaraldehyde and 0.01% v/v Triton X-100 in 0.1M phosphate buffer (pH 7.2) by vacuum infiltration (10 kPa).
  • Sections were washed in PBS for 10 min, incubated in 0.1M hydroxyl-ammoniumchloride for 30 min and washed again in PBS for 5 min. Sections were blocked in 0.5% w/v Bovine Serum Albumin Fraction V (Sigma- Aldrich) for 30 min and rinsed twice with PBS for 15 min. Samples were incubated overnight at 4 °C in anti-PRIl -FITC diluted 1 :600 or 1 : 1200 in PBS, washed twice for 15 min and twice for 30 min in PBS. The sections were finally mounted on slides in glycerol Citifluor AF2 for microscopic observation. For vitality assessments liquid grown bacteria were stained with propidium iodide (Sigma) at a final concentration of 5 ⁇ for 5 min in the dark.
  • P. s. pv. aesculi PD4818 and its pMP4655 harbouring derivative were cultured at 20 °C in either LB or minimal medium containing 20 mM NaCl, 20 mM phosphate buffer, 10 mM (NH 4 ) 2 S0 4 , 5 mM MgS0 4 , 10 mM fructose and 10 mM mannitol (pH 6.1) [50,51]. After 2-3 days, cultures were observed by fluorescence microscopy as described above or prepared for scanning electron microscopy by fixing 1 mL samples with 3% v/v glutaraldehyde dissolved in the appropriate medium.
  • Cultures of 125 mL of P. s. pv. aesculi PD4818 were either grown in duplo in minimal medium for 5 days or in minimal medium for 3 days and then under salt stress (0.4 M NaCl in minimal medium) for 2 days. Cultures were centrifuged for 15 min (10,000 x g, RT) and three volumes of ice cold ethanol were added to the supernatant to precipitate the extracellular material o/n at 4 °C. The precipitate was collected after centrifugation (30 min, 10,000 x g, 4 °C) and subsequently dialyzed o/n (Medicell Visking, MWCO 12,000-14,000 Da) against dH 2 0.
  • the amount of uronic acids was determined using the m-hydroxydiphenyl assay as described by Blumenkrantz and Asboe-Hansen [52] and galacturonic acid was used as standard.
  • the sugar composition was determined in more detail by High Performance Anion Exchange Chromatography (HPAEC) using an ICS-3000 Ion Chromatography HPLC system equipped with a CarboPac PA-1 column (2 x 250 mm) in combination with a CarboPac PA guard column (2 x 25 mm) and a pulsed electrochemical detector in pulsed amperometric detection mode (Dionex). A flow rate of 0.3 mL min 1 was used and the column was equilibrated with 16 mM NaOH.
  • HPAEC High Performance Anion Exchange Chromatography
  • EXAMPLE 1 Lignin and suberin are deposited near the wound boundary
  • bacteria were applied to small wounds inflicted on the stems of A. hippocastanum seedlings. The method of wounding did not cause irrecoverable damage, since mock inoculated wounds showed generation of a new periderm confluent with the existing one within 3 months (Fig. IB upper panel). Inoculated wounds did not exhibit such a recovery (Fig. IB lower panel).
  • lignin is a major component of the early wound repair responses in A. hippocastanum bark.
  • lignin another histological compound with autofluorescent properties that could potentially be of importance in the barrier lining the wound is suberin [13,14,17].
  • Sudan IV stained sections were observed under fluorescence conditions whereupon suberin lamellae appeared in dim red, while lignin autofluorescence was unaltered (See materials and methods).
  • Fig. 1E/F From the sixth day after inoculation and onwards suberin was found as a lamella against the cell wall of the cells that exhibited lignification earlier (Fig. 1E/F). The deposition was uniform and did not occlude pit fields (Fig. 1G), supporting that the lining of lignified and suberized cells around the wound perimeter functions in sealing off the wound from healthy tissues.
  • the effectiveness of the cellular response in restraining pathogen invasion after wounding can be assessed when related to observations on bacterial proliferation.
  • immunolabelling the spread of inoculated P. s. pv. aesculi was monitored. During the first 2 days after inoculation, the bacteria were still mainly localized on the wound surface, with some individual bacteria present several cell layers deeper (data not shown). At the fourth day after inoculation several scattered bacteria were found behind the developing barrier zone. These first signs of bacterial ingress were clearly discernible in samples taken 6 days post inoculation, in which small clusters of bacterial cells were found in intercellular spaces of parenchyma cells, evidently inward from the lignified and suberized lining (Fig. 2A, arrowheads). Besides the colonization of living tissues, bacterial clusters remained in the wound fissure (Fig. 2 A, arrows).
  • EXAMPLE 4 P. syringae pv. aesculi is embedded in an extracellular matrix in planta
  • alginate was the predominant polysaccharide in the extracellular material produced by P. s. pv. aesculi under salt stress.
  • Previous studies point to the possible additional presence of glucuronic acid [22] which elutes approximately at the same time as mannuronic acid.
  • glucuronic acid [22] which elutes approximately at the same time as mannuronic acid.
  • rhamnose and glucose although at lower concentrations than alginate, were identified (Fig. 6). Minor traces of an unidentified sugar were also present.
  • Alginate is often found to be produced by plant pathogenic Pseudomonas spp. when associated with their host [23-25]. Its secretion aids in retaining water and is thereby thought to contribute to bacterial colonization and symptom development [26].
  • the ability of P. s. pv. aesculi to produce alginate was further supported by the presence of genes associated with its biosynthesis and regulation thereof as screened in silico. Sequence data of P. s. pv. aesculi strain 2250 [8] indicated that this strain has a full repertoire of genes encoding alginate biosynthesis and regulatory proteins with close homology to these of P. s. pv. tomato DC3000 (Table IB). Furthermore, most of the genes predicted to function in biosynthesis were found to be arranged in the canonical alginate biosynthesis operon structure [27].
  • Alg8 alginate biosynthesis protein Alg8 ZP " 06478556 493 97%
  • Alg44 alginate biosynthesis protein Alg44 ZP " 06478557 390 92%
  • AlgE alginate biosynthesis protein AlgE ZP " 06478559 493 97%
  • AlgG alginate biosynthesis protein AlgG ZP " 06478560 536 92%
  • AlgX alginate biosynthesis protein AlgX ZP " 06478561 479 97%
  • Algl alginate biosynthesis protein Algl ZP " 06478563 518 97%
  • AlgJ alginate biosynthesis protein AlgJ ZP " 06478564 391 95%
  • AlgQ anti-R A polymerase sigma 70 ZP 06477758 157 96%
  • the barrier sometimes seemed a bit more faint near invading bacteria, hinting at pathogen-mediated degeneration (Fig. 2A). This could be facilitated by the various pathways for degradation of lignin-related compounds uncovered in the P. s. pv aesculi genome [8]. Although not determinative in restraining P. s. pv. aesculi invasion, presence of the observed barrier might still serve a beneficial, protective role. For instance, it could prevent water and mineral flow towards the bacteria, prevent diffusion of bacterial toxins and effectors to healthy plant tissues, reduce tissue digestibility and give rigidity, preventing further tissue collapse.
  • Fig. 3D The bacterial cells found near the forefront of expanding infections were small and densely clustered.
  • disease symptoms on the cellular level included occasional colonization of single phloem/cortex parenchyma cells by a large bacterial cluster (Fig. 2B, J).
  • Fig. 2B, J The bacterial accumulations might be important in the infection process, since expression of pathogenic traits often requires a certain, quorum sensing coordinated, cell density [32]. That such a quorum sensing system may act within the observed bacterial clusters in horse chestnut tissues is supported by the quorum size described for P. syringae (down to -20 individuals [33]) and the ability of Indian P. s. pv. aesculi to produce N-acyl- homoserine lactones [34].
  • the observed matrix may play a direct role in pathogenicity.
  • One such function may be the suppression of host defence
  • thermal treatment may be a feasible, non-invasive control method employable for disease management on a larger scale.
  • a potential issue with applying heat-treatment under field conditions could lie in differences in temperature sensitivity that may occur among various isolates, as is reported for Erwinia amylovora [44].
  • the extremely high genetic homogeneity among UK P. s. pv. aesculi isolates [8] implies that variation in heat-resistance among bacterial strains is minimal, suggesting heat-treatment of A. hippocastanum may be a widely applicable method.
  • A Typical symptoms of P. syringae pv. aesculi associated bleeding disease observed on the trunk of an A. carnea tree, including bleeding of amber coloured sap and cracking of the bark (arrowheads). Photo taken in Sept. 2008 at N51° 57' 26"; E5° 34' 10".
  • B Appearance of surface wounds on A. hippocastanum seedlings mock-inoculated (top panel) or inoculated with P. s. pv. aesculi PD5126 (bottom panel) after 3 months. While the seedling bark recovers when mock-inoculated by regeneration of periderm, the wounded tissue appears blackened and sunken when inoculated with P. s. pv. aesculi.
  • L-N Longitudinal sections of the cambium region with bacteria in intercellular spaces of phloem parenchyma (L), ray parenchyma (M) and along phloem fibres (N), 3 weeks after inoculation.
  • O P. s. pv. aesculi in intercellular spaces observed in a transversal section of the cambial zone at 4 weeks after inoculation.
  • R-T Transversal sections of enclosures surrounded by periderm with bacteria ex planta at 4, 10 and 18 weeks after inoculation respectively.
  • Figure 3 Behaviour of GFP-expressing P. syringae in planta
  • A An A. hippocastanum seedling inoculated with strain PD4818-pMP4655 photographed after 2 months shows vertical lesion expansion (up to 6 cm in length) and tissue necrosis after removal of the outer tissues of the bark. The inoculation site is indicated with an arrowhead. Note the bending of the infection around the lower node. The scale bar indicates 1 cm.
  • Figure 4 A fibrillar matrix produced in vitro by P. s. pv. aesculi enwraps aggregates of immobilized bacteria.
  • FIG. 5 Growth of two Pseudomonas syringae pv. aesculi strains and their GFP-expressing derivatives. Overnight cultures of P. s. pv. aesculi PD4818, PD5126 and their derived transformants harbouring pMP4655 were diluted to an OD 6 oo of 0.005 in fresh LB. The optical density at 600 nm of the cultures was recorded during growth at 28 °C and 180 rpm. The presented values are means ( ⁇ standard deviation) of three replicates and an asterisk indicates significant differences as determined with Students 2-tailed t-test (P ⁇ 0.001).
  • FIG. 6 High performance anion exchange chromatography of acid hydro lysed extracellular polysaccharide of Pseudomonas syringae pv. aesculi PD4818 cultivated without (A) and with salt stress (B) and acid hydrolysed alginate from Laminaria hyperborea (C) and Macrocystis pyrifera (D). Each subsequent plot has been shifted 2.4 minutes for clarity.
  • the marked peaks represent: I, Rhamnose; II, Unidentified compound; III, Glucose; IV, Degradation pattern of alginate (guluronic acid and mannuronic acid).
  • Figure 7 shows a young A. hippocastanum branch undergoing heat treatment in a climate chamber.
  • Figure 8 shows a mature A. hippocastanum tree undergoing heat treatment outside using hoses transporting water at 40°C, wrapped around the trunk.
  • Woodward S, Pocock S (1996) Formation of the ligno-suberized barrier zone and wound periderm in four species of European broad-leaved trees. European Journal of Forest Pathology 26: 97-105.

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

L'invention concerne le traitement thermique non destructif d'arbres pour arrêter la progression de maladies. Certaines espèces bactériennes sont des pathogènes majeurs des arbres. L'établissement, la croissance et la qualité des arbres peuvent être affectés par l'apparition de ces maladies. Les arbres infectés présentent une nécrose importante du phloème et du cambium, qui peut conduire au final à un dépérissement terminal. L'emplacement endophytique et la capacité de ces pathogènes à créer une matrice protectrice les rend difficilement accessibles aux agents de lutte. La présente invention concerne des procédés et des appareils pour lutter contre ou stopper des infections bactériennes dans des arbres par un traitement thermique comprenant l'incubation de plantes ou de parties de plantes à environ 39 °C pendant une durée non inférieure à 48 heures.
PCT/EP2013/063567 2012-06-29 2013-06-27 Traitement thermique non destructif d'arbres pour arrêter la progression de maladies WO2014001476A2 (fr)

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EP13732160.0A EP2866550A2 (fr) 2012-06-29 2013-06-27 Traitement thermique non destructif d'arbres pour arrêter la progression de maladies

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GBGB1211638.0A GB201211638D0 (en) 2012-06-29 2012-06-29 Heat treatment of tree and shrubs to stop or ameliorate progression of bacterial diseases
GB1211638.0 2012-06-29
GB1211710.7 2012-07-02
GBGB1211710.7A GB201211710D0 (en) 2012-07-02 2012-07-02 Heat treatment of trees and shrubs to stop or ameliorate progression of bacterial diseases

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KR101577529B1 (ko) 2014-04-07 2015-12-17 순천대학교 산학협력단 참다래 궤양병균 검출용 키트 및 상기 키트를 이용한 참다래 궤양병의 진단방법
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FR35295E (fr) * 1928-01-17 1929-12-31 Appareil pour échauder les vignes, arbres et autres plantes
WO2006021225A1 (fr) * 2004-08-24 2006-03-02 Hartwig Pollinger Procede et dispositif pour traiter des chenes-lieges

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