WO1986000934A1 - Method for the early determination of plant disease - Google Patents

Method for the early determination of plant disease Download PDF

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Publication number
WO1986000934A1
WO1986000934A1 PCT/DE1985/000251 DE8500251W WO8600934A1 WO 1986000934 A1 WO1986000934 A1 WO 1986000934A1 DE 8500251 W DE8500251 W DE 8500251W WO 8600934 A1 WO8600934 A1 WO 8600934A1
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Prior art keywords
differences
plants
nucleic acid
detection
healthy
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PCT/DE1985/000251
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German (de)
French (fr)
Inventor
Jürgen SCHUMACHER
Detlev Riesner
Original Assignee
Schumacher Juergen
Detlev Riesner
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Publication of WO1986000934A1 publication Critical patent/WO1986000934A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the invention is a method in which the nucleic acid extracts from healthy and diseased plants are compared with one another and the health status of the plants can be deduced from the presence or absence of certain nucleic acid molecules.
  • Infectious diseases are becoming more and more serious in the field of agriculture. This is, among other things, a consequence of the cultivation of highly refined varieties in monocultures.
  • Current examples are fungal diseases in cereals, viral diseases in potatoes, sugar beet and fruit trees. It is in order to carry out damage surveys for forest diseases and to obtain information about how the disease spreads necessary to check the condition of the trees at certain time intervals.
  • the tree stands are examined by forest officials and divided into three damage levels. Criteria for the assessment are, for example, the needling and leafing of the crowns, discoloration of needles and leaves, so-called anxiety drives and changes in trunk and bark.
  • the symptom expression is only clear in very few cases, the division into the Subjective stages take place subjectively.
  • the subject of the presented invention is a gel electrophoretic method, which among other things enables a reliable distinction between sick and healthy trees to be made very early.
  • a gel electrophoretic examination is shown by way of example in FIG. 1.
  • 1 g each of pine trees was obtained from an apparently healthy or sick tree in a Tris-HCL buffer at pH 8.0 and an ionic strength of 100 mM in the presence of 25th SDS and 25 M mercaptoethanol together with phenol and chloroform are homogenized in an Ultra Turrax. After filtration and re-extraction with phenol / chloroform, the nucleic acid was precipitated from the plant extract by adding ethanol. The precipitate was dissolved in 200 ul electrophoresis buffer.
  • the nucleic acids were separated in a two-dimensional 5% polyacrylamide gel electrophoresis, initially under denaturing conditions at 8 M urea and 50 ° C. and then in the second dimension -3- ran at 20 ° C without urea. The gel was then stained with silver nitrate.
  • Fig. 1b in der_ sample of the diseased tree indicated by the arrow 'band labeled zusäzlich to the bands of the sample of the healthy tree (Fig. 1a) seen surprisingly turned out that these additional nucleic acids place independently of the Stand ⁇ and Age of the pines in all trees affected by "forest death" can be proven.
  • Significant differences in the ⁇ -sand pattern after two-dimensional gel electrophoresis have now also been found in diseased spruce and beech trees.
  • the method can be applied to the diagnosis of diseases of agricultural crops.
  • diseases of agricultural crops examples include viral diseases that occur in cereals, potatoes, sugar beets, fruit trees and grapevines.
  • the method described has hitherto been used successfully in the Rizomania disease of sugar beet, which is caused by the Beet Necrotic Yellow Vein Virus (BNYVV), and in the vein yellowing of apples, which is caused by unidentified viruses .
  • BNYVV Beet Necrotic Yellow Vein Virus
  • the additional nucleic acid band caused by the disease is already visible in the one-dimensional gel electrophoresis. The result is confirmed by Fig. 2; the additional band in the sample of the sick plant (left side) compared to the sample of the healthy plant (right side) is marked by an arrow.
  • aqueous two-phase system With the help of the aqueous two-phase system mentioned, the extraction of the nucleic acids can be carried out with great advantage.
  • an aqueous two-phase system has proven to be suitable, which consists on the one hand of an aqueous solution of a neutral salt and on the other hand of an aqueous polyethylene glycol solution, the nucleic acids accumulating in the other, ie the brine phase.
  • the Zu ⁇ by administration of ethanol precipitated nucleic acids in 8 parts of H.-0 and treated with 21 parts of 30% (w / w) potassium phosphate, and ' 11 parts 30% (w / w) polyethylene glycol 6000 mixed.
  • the solution is briefly centrifuged to separate the phases.
  • the nucleic acids are quantitatively in the lower, the salt phase, from which they are precipitated on ice with 0.1% cetyltrimethylammonium bromide (CTAB) for 30 min.
  • CTAB cetyltrimethylammonium bromid
  • Cetyltrimethylammonium bromide precipitate is washed twice with 70% ethanol, dried and taken up in H_0.
  • the cleaning of the nucleic acids from the interfering plant constituents is so effective that differences in the nucleic acid pattern are already visible in the one-dimensional gel electrophoresis (see FIG. 2).
  • the use of extraction with the aid of the aqueous two-phase system saves additional cleaning procedures in all subsequent steps.
  • RNA or nucleic acid sequence it is entirely possible to carry out the tests for a specific RNA or nucleic acid sequence not only by gel electrophoresis, but also with the aid of modern molecular hybridization techniques. If such a nucleic acid is found in leaf or needle samples, such as the additional RNA in pine tissue in the example presented, it can be assumed that the tree is diseased, even if there has not yet been any visible symptom manifestation.
  • the test thus enables objective early detection and mapping of the damage. Trees can be felled in good time before the wood quality has decreased due to the disease. Resistant or more resistant trees can be recognized early.
  • the test is also very easy and quick to carry out and is suitable for serial examinations of a few hundred trees per person per examiner.

Abstract

It can be proved that typical modifications exist in samples of nucleic acid of ill plants. Independently from the origin location and the edge of the plants, additional bands appear during the bidimensional gel-electrophoretic separation of nucleic acids. Said supplementary nucleic acids may be tested, for example with molecular hybridation technics. Early objective diagnoses may thus be obtained, which enable to take on time the required measures to combat the disease, as well as to establish the mapping of large extensions of damages caused to plants. The test is very simple and is adapted to investigation in series.

Description

Verfahren zur früherkeπnung von PflanzenkrankheitenProcess for the early diagnosis of plant diseases
Bei der Erfindung handelt es sich um ein Verfahren, bei dem die Nuklein- säureextrakte aus gesunden und kranken Pflanzen miteinander verglichen werden und aus dem Auftreten oder der Abwesenheit bestimmter Nuklein- säuremoleküle auf den Gesundheitszustand der Pflanzen geschlossen werden kann.The invention is a method in which the nucleic acid extracts from healthy and diseased plants are compared with one another and the health status of the plants can be deduced from the presence or absence of certain nucleic acid molecules.
Die Früherkennung von Pflanzenkrankheiten ist von großer wirtschaft¬ licher Bedeutung für die Land- und Forstwirtschaft. In den letzten zehn Jahren wurden in Mitteleuropa zunehmend neuartige Erkrankungen an Wald¬ bäumen beobachtet, die unter dem Begriff "Waldsterben" bekannt geworden sind. Trotz verstärkter Forschungen auf diesem Gebiet ist die Ursache der Erkrankungen und damit natürlich eine mögliche Therapie nicht ein¬ deutig bekannt. Die Hauptschuld am Waldsterben wird zur Zeit der Luft- Verschmutzung gegeben. Hauptsächlich der saure Regen sowie hohe Konzen¬ trationen an Ozon und Stickoxyden werden für die Erkrankungen verant¬ wortlich gemacht. In zunehmendem Maße werden auch Infektionseinflüsse z.B. durch Pilze und Viren als mögliche Ursachen diskutiert. Die Verluste durch Waldschäden wachsen mittlerweile in erschreckende Höhe. Von den insgesamt über 7 Millionen Hektar Waldfläche in der Bundesrepublik sind inzwischen etwa 40% geschädigt, das entspricht einem Vermögenswert von ungefährt 70 Milliarden DM. Die Tendenz ist weiter steigend. Damit sind auch die Arbeitsplätze der 800.000 in der Fo st¬ wirtschaft Beschäftigten gefährdet. In absehbarer Zeit wird kein gesun- der Baum mehr gefällt werden können, da zunächst die erkrankten Bäume herausgeschlagen werden müssen. Die Auswirkungen dieser Entwicklung auf die Holzwirtsc aft sind noch nicht abzusehen.The early detection of plant diseases is of great economic importance for agriculture and forestry. In the past ten years, new diseases in forest trees have increasingly been observed in Central Europe, which have become known under the term "forest deaths". Despite intensified research in this area, the cause of the diseases and thus of course a possible therapy is not clearly known. The main culprit for forest death is given at the time of air pollution. Mainly the acid rain and high concentrations of ozone and nitrogen oxides are made responsible for the diseases. Influences of infection, e.g. discussed by fungi and viruses as possible causes. The losses caused by forest damage are growing at an alarming rate. Around 40% of the more than 7 million hectares of forest in the Federal Republic have been damaged, which corresponds to an asset of approximately DM 70 billion. The trend is rising. The jobs of the 800,000 people employed in the forestry sector are also at risk. In the foreseeable future, it will no longer be possible to fell a healthy tree, because the diseased trees must first be cut out. The effects of this development on the timber industry cannot yet be predicted.
Im Bereich der Landwirtschaft werden Infektionskrankheiten immer schwer¬ wiegender. Dies ist unter anderem eine Folge des Anbaus hoch veredelter Sorten in Monokulturen. Aktuelle Beispiele sind Pilzkrankheiten bei Ge¬ treide, Viruskrankheiten bei Kartoffel, Zuckerrübe und Obstbäumen. Um Schadenserhebungen bei Walderkrankungen durchzuführen sowie Aussagen über Art und Weise der Ausbreitung der Krankheit zu erhalten, ist es notwendig, in bestimmten Zeitintervallen den Zustand der Bäume zu über¬ prüfen. Hierzu werden die Baumbestände von Forstbeamten in Augenschein genommen und in drei Schadstufen eingeteilt. Kriterien für die Beur¬ teilung stellen dabei z.B. der Benadelungs- und Belaubungszustand der Kronen, Verfärbung von Nadeln und Blättern, sogenannte Angsttriebe sowie Veränderungen von Stamm und Rinde dar. Da die Symptomausprägung aber nur in den wenigsten Fällen eindeutig ist, muß die Einteilung in die Schad¬ stufen subjektiv erfolgen. Auch eine Beurteilung des Wald zu Standes, aus der Luft mit Hilfe von Infrarotaufnahmen ist nicht ausreichend. Jüngere Bäume können mit dieser Technik z.B. nicht beurteilt werden. Gerade bei solchen Bäumen wäre eine objektive Beurteilung wünschenswert. Könnte man bereits bei Setzlingen erkennen, ob sie erkrankt sind, so könnte man sie rechtzeitig durch gesunde Pflanzen ersetzen ohne weiteren nutzlosen Kul¬ tivierungsaufwand. Die Diagnose von Krankheiten bei landwirtschaftlichen Nutzpflanzen kann durch die Beurteilung der Symptomausprägung an den Pflanzen direkt oder nach Übertragung der Krankheit auf Testpflanzeπ erfolgen. Dieses Ver¬ fahren ist langwierig und subjektiven Einflüssen unterworfen. Durch ge¬ schultes Personal unter Verwendung von erheblichem apparativen Aufwand wie Mikroskop oder Elektronenmikroskop sind Diagnosen auch möglich. Die neueren immunologischen Verfahren, die hauptsächlich bei Viruserkrankun¬ gen Anwendung finden, setzen eine gute Charakterisierung der Krankheits¬ erreger voraus und werden oft durch die Pflanzeninhaltsstoffe in ihrer Aussagekraft stark beeinträchtigt. Gegenstand der vorgestellten Erfindung ist ein gelelektrophoretisches Verfahren, womit unter anderem eine sichere Unterscheidung zwischen kranken und gesunden Bäumen sehr frühzeitig möglich ist. Eine solche gelelektrophoretische Untersuchung ist beispielhaft in Figur 1 darge¬ stellt. Hierzu wurden jeweils 1 g Kiefernna,deln von einem augenschein- lieh gesunden bzw. kranken Baum in einem Tris-HCL Puffer bei pH 8,0 und einer Ionenstärke von 100 mM in Gegenwart von 25. SDS und 25 M Mercapto- ethanol zusammen mit Phenol und Chloroform in einem Ultra Turrax homo¬ genisiert. Nach Filtration und Reextraktion mit Phenol /Chloroform wurde die Nukleinsäure aus dem Pflanzenextrakt durch Zugabe von Ethanol ge- fällt. Das Präzipitat wurde in 200 μl Elektrophoresepuffer gelöst. Die Auftrennung der Nukleinsäuren erfolgte in einer zweidi ensionalen 5% Polyacrylamid-Gelelektrophorese, wobei sie zunächst unter denaturieren¬ den Bedingungen bei 8 M Harnstoff und 50°C und dann in der 2. Dimension -3- ohne Harnstoff bei 20°C liefen. Das Gel wurde anschließend mit Silber- Nitrat gefärbt. Deutlich ist in der_ Probe des erkrankten Baumes (Fig. 1b) die durch den Pfeil markierte 'Bande zusäzlich zu den Banden der Probe des gesunden Baumes (Fig. 1a) erkennbar, überraschenderweise zeigte sich, daß diese zusätzlichen Nukleinsäuren unabhängig vom Stand¬ ort und Alter der Kiefern in allen vom "Waldsterben" betroffenen Bäumen nachgewiesen werden können. Ebenfalls deutliche Unterschiede im -ßanden- uster nach der zweidimensionalen Gelelektrophorese konnten inzwischen auch bei erkrankten Fichten und Buchen gefunden werden.Infectious diseases are becoming more and more serious in the field of agriculture. This is, among other things, a consequence of the cultivation of highly refined varieties in monocultures. Current examples are fungal diseases in cereals, viral diseases in potatoes, sugar beet and fruit trees. It is in order to carry out damage surveys for forest diseases and to obtain information about how the disease spreads necessary to check the condition of the trees at certain time intervals. For this purpose, the tree stands are examined by forest officials and divided into three damage levels. Criteria for the assessment are, for example, the needling and leafing of the crowns, discoloration of needles and leaves, so-called anxiety drives and changes in trunk and bark. However, since the symptom expression is only clear in very few cases, the division into the Subjective stages take place subjectively. An assessment of the state of the forest from the air with the help of infrared images is also not sufficient. Younger trees, for example, cannot be assessed using this technique. With such trees in particular, an objective assessment would be desirable. If seedlings could already be recognized as to whether they were diseased, they could be replaced in good time by healthy plants without any further useless cultivation effort. The diagnosis of diseases in agricultural crop plants can be carried out by assessing the symptoms of the plants directly or after the disease has been transferred to test plants. This process is lengthy and subject to subjective influences. Diagnoses are also possible by trained personnel using considerable equipment such as a microscope or electron microscope. The newer immunological methods, which are mainly used for viral diseases, require a good characterization of the pathogens and are often severely impaired in their informative value by the plant ingredients. The subject of the presented invention is a gel electrophoretic method, which among other things enables a reliable distinction between sick and healthy trees to be made very early. Such a gel electrophoretic examination is shown by way of example in FIG. 1. For this purpose, 1 g each of pine trees was obtained from an apparently healthy or sick tree in a Tris-HCL buffer at pH 8.0 and an ionic strength of 100 mM in the presence of 25th SDS and 25 M mercaptoethanol together with phenol and chloroform are homogenized in an Ultra Turrax. After filtration and re-extraction with phenol / chloroform, the nucleic acid was precipitated from the plant extract by adding ethanol. The precipitate was dissolved in 200 ul electrophoresis buffer. The nucleic acids were separated in a two-dimensional 5% polyacrylamide gel electrophoresis, initially under denaturing conditions at 8 M urea and 50 ° C. and then in the second dimension -3- ran at 20 ° C without urea. The gel was then stained with silver nitrate. Significantly (Fig. 1b) in der_ sample of the diseased tree indicated by the arrow 'band labeled zusäzlich to the bands of the sample of the healthy tree (Fig. 1a) seen surprisingly turned out that these additional nucleic acids place independently of the Stand¬ and Age of the pines in all trees affected by "forest death" can be proven. Significant differences in the β-sand pattern after two-dimensional gel electrophoresis have now also been found in diseased spruce and beech trees.
In ähnlicher Weise kann das Verfahren auf die Diagnose von Krankheiten landwirtschaftlicher Nutzpflanzen angewendet werden. Beispiele hierfür sind Viruskrankheiten, welche bei Getreide, Kartoffel, Zuckerrüben, Obstbäumen und Weinreben auftreten. Das beschriebene Verfahren wurde bisher erfolg¬ reich bei der Rizomania-Krankheit von Zuckerrüben, die durch das Beet Necrotic Yellow Vein Virus (BNYVV) hervorge¬ rufen wird, und bei der Adernvergilbung von Äpfeln, die durch nicht charakterisierte Viren hervorgerufen wird, an- gewendet. Durch das weiter unten beschriebene Verfahren der Extraktion mit Hilfe eines wässrigen Zweiphasensystems ist die durch die Krankheit verursachte zusätzliche Nukleinsäu- rebande bereits in der eindimensionalen Gelelektrophorese sichtbar. Das Ergebnis ist durch Fig. 2 belegt; die zusätz- liehe Bande in der Probe der kranken Pflanze (linke Seite) im Vergleich zur Probe der gesunden Pflanze (rechte Seite) ist durch einen Pfeil markiert.Similarly, the method can be applied to the diagnosis of diseases of agricultural crops. Examples include viral diseases that occur in cereals, potatoes, sugar beets, fruit trees and grapevines. The method described has hitherto been used successfully in the Rizomania disease of sugar beet, which is caused by the Beet Necrotic Yellow Vein Virus (BNYVV), and in the vein yellowing of apples, which is caused by unidentified viruses . By means of the method of extraction using an aqueous two-phase system described below, the additional nucleic acid band caused by the disease is already visible in the one-dimensional gel electrophoresis. The result is confirmed by Fig. 2; the additional band in the sample of the sick plant (left side) compared to the sample of the healthy plant (right side) is marked by an arrow.
Mit Hilfe des erwähnten wässrigen Zweiphasensystems kann die Extraktion der Nukleinsäuren mit großem Vorteil durch¬ geführt werden. Insbesondere hat sich ein solches wässriges Zweiphasensystem als geeignet erwiesen, das einerseits aus einer wässrigen Lösung eines neutralen Salzes und anderer¬ seits aus einer wässrigen Polyethylenglykollösung besteht, wobei sich die Nukleinsäuren in der anderen, also der Sole¬ phase ansammeln. Beispielsweise werden dazu die durch Zu¬ gabe von Ethanol gefällten Nukleinsäuren in 8 Teilen H.-0 gelöst und mit 21 Teilen 30 % (w/w) Kaliumphosphat und ' 11 Teilen 30 % (w/w) Polyethylenglykol 6000 gemischt. Zur Phasentrennung wird die Lösung kurz zentrifugiert. Die Nuk¬ leinsäuren befinden sich quantitativ in der unteren, der Salzphase, aus der sie mit 0,1% Cetyltrimethylammonium- bromid (CTAB) für 30 min auf Eis ausgefällt werden. DerWith the help of the aqueous two-phase system mentioned, the extraction of the nucleic acids can be carried out with great advantage. In particular, such an aqueous two-phase system has proven to be suitable, which consists on the one hand of an aqueous solution of a neutral salt and on the other hand of an aqueous polyethylene glycol solution, the nucleic acids accumulating in the other, ie the brine phase. For example, the Zu¬ by administration of ethanol precipitated nucleic acids in 8 parts of H.-0 and treated with 21 parts of 30% (w / w) potassium phosphate, and ' 11 parts 30% (w / w) polyethylene glycol 6000 mixed. The solution is briefly centrifuged to separate the phases. The nucleic acids are quantitatively in the lower, the salt phase, from which they are precipitated on ice with 0.1% cetyltrimethylammonium bromide (CTAB) for 30 min. The
Cetyltrimethylammoniumbromid-Niederschlag wird zweimal mit 70-igem Ethanol gewaschen, getrocknet und in H_0 aufgenom¬ men. Die Reinigung der Nukleinsäuren von den störenden Pflanzeninhaltsstoffen ist dabei so effektiv, daß Unter- schiede im Nukleinsäuremuster bereits in der eindimensiona¬ len Gelelektrophorese sichtbar werden (s. Fig. 2). Die Ver¬ wendung der Extraktion mit Hilfe des wässrigen Zweiphasen¬ systems erspart bei allen Folgeschritten zusätzliche Reini¬ gungsprozeduren.Cetyltrimethylammonium bromide precipitate is washed twice with 70% ethanol, dried and taken up in H_0. The cleaning of the nucleic acids from the interfering plant constituents is so effective that differences in the nucleic acid pattern are already visible in the one-dimensional gel electrophoresis (see FIG. 2). The use of extraction with the aid of the aqueous two-phase system saves additional cleaning procedures in all subsequent steps.
Es ist durchaus möglich, die Untersuchungen auf eine bestimmte RNA bzw. Nukleinsäuresequenz nicht nur gelelektrophoretisch, sondern auch mit Hilfe moderner molekularer Hybridisierungstechniken durchzuführen. Findet sich dann in Blatt- oder Nadelproben eine solche Nukleiπsäure, wie im vorgestellten Beispiel die zusätzliche RNA in Kieferngewebe, so kann davon ausgegangen werden, daß der Baum erkrankt ist, auch wenn eine sichtbare Symptomausprägung noch nicht stattgefunden hat. Damit erlaubt der Test eine objektive Früherkennung und Kartierung der Schäden. Bäume können rechtzeitig gefäLlt werden, bevor eine Minderung der Holzquali- t t, bedingt durch die Krankheit, eingesetzt hat. Resistente oder resi- stentere Bäume können frühzeitig erkannt werden. Der Test ist zudem sehr einfach und schnell durchzuführen und eignet sich für Reihenunter¬ suchungen einiger Hundert Bäume pro Tag pro untersuchende Person. It is entirely possible to carry out the tests for a specific RNA or nucleic acid sequence not only by gel electrophoresis, but also with the aid of modern molecular hybridization techniques. If such a nucleic acid is found in leaf or needle samples, such as the additional RNA in pine tissue in the example presented, it can be assumed that the tree is diseased, even if there has not yet been any visible symptom manifestation. The test thus enables objective early detection and mapping of the damage. Trees can be felled in good time before the wood quality has decreased due to the disease. Resistant or more resistant trees can be recognized early. The test is also very easy and quick to carry out and is suitable for serial examinations of a few hundred trees per person per examiner.

Claims

P A T E N T A N S P R Q C H EP A T E N T A N S P R Q C H E
5 1. Verfahren zur Erkennung von Pflanzenkrankheiten, dadurch gekenn¬ zeichnet, daß Unterschiede im Nukleinsäuremuster zwischenkrankeπ und gesunden Pflanzen zur Erkennung ausgenutzt werden.5 1. Method for the detection of plant diseases, characterized gekenn¬ characterized in that differences in the nucleic acid pattern between sick and healthy plants are used for detection.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß es zur Erken- 0 nung von Waldschäden dient und Unterschiede im Nukleinsäuremuster zwischen kranken und gesunden Bäumen zur Erkennung ausgenutzt werden.2. The method according to claim 1, characterized in that it is used for Erken- 0 detection of forest damage and differences in the nucleic acid pattern between sick and healthy trees are used for detection.
3. Verfahren nach den Ansprüchen 1-2, dadurch gekennzeichnet, daß die 15 Unterschiede vor Ausprägung der Krankheitssymptome ausgenutzt werden können.3. The method according to claims 1-2, characterized in that the 15 differences can be exploited before the symptoms of the disease are manifested.
4. Verfahren nach den Ansprüchen 1-3, dadurch gekennzeichnet, daß die Unterschiede gelelektrophoretisch nachgewiesen werden können. 04. The method according to claims 1-3, characterized in that the differences can be detected by gel electrophoresis. 0
5. Verfahren nach den Ansprüchen 1-4, dadurch gekennzeichnet, daß die Unterschiede mit Hilfe einer zweidimensionalen Gelelektrophorese in Verbindung mit einer Silberfärbung nachgewiesen werden können.5. The method according to claims 1-4, characterized in that the differences can be detected with the aid of a two-dimensional gel electrophoresis in connection with a silver stain.
256. Verfahren nach den Ansprüchen 1-3, dadurch gekennzeichnet, daß die Unterschiede mit Hilfe molekularer Hybridisieruπgstechniken nachge¬ wiesen werden können.256. Method according to claims 1-3, characterized in that the differences can be demonstrated using molecular hybridization techniques.
7. Verfahren nach den Ansprüchen 1-3 und 6, dadurch gekennzeichnet, daß 30 die Unterschiede mit Hilfe der Dot-Blot-Hybridisierung nachgewiesen werden können.7. The method according to claims 1-3 and 6, characterized in that 30 the differences can be detected with the aid of dot-blot hybridization.
8. Verfahren nach den Ansprüchen 1-2, 6 und 7, dadurch gekennzeichnet, daß die zur Anwendung ko mmende Hybridisierungsprobe die komplemen-8. The method according to claims 1-2, 6 and 7, characterized in that the hybridization sample coming to use the complementary
35 täre Sequenz einer der Banden darstellt, die in der kranken Pflanze anwesend, in der gesunden Pflanze abwesend ist. Verfahren zur Extraktion von Nukleinsäure aus Pflanzen, insbesondere für ein Verfahren nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, daß ein wässriges Zwei¬ phasensystem benutzt wird. 35 tary sequence represents one of the bands present in the diseased plant and absent in the healthy plant. Process for the extraction of nucleic acid from plants, in particular for a process according to one of claims 1 to 8, characterized in that an aqueous two-phase system is used.
PCT/DE1985/000251 1984-08-01 1985-07-25 Method for the early determination of plant disease WO1986000934A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DEP3428386.2 1984-08-01
DE3428386 1984-08-01
DE19853503978 DE3503978A1 (en) 1984-08-01 1985-02-06 METHOD FOR THE EARLY DETECTION OF PLANT DISEASES
DEP3503978.7 1985-02-06

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0349024A1 (en) * 1988-05-02 1990-01-03 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno A method for the simultaneous determination of DNA sequence variations at a large number of sites, and a kit therefor

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Publication number Priority date Publication date Assignee Title
EP0349024A1 (en) * 1988-05-02 1990-01-03 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno A method for the simultaneous determination of DNA sequence variations at a large number of sites, and a kit therefor

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EP0191059A1 (en) 1986-08-20
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