WO2000019204A1 - METHOD OF DETECTING $i(PLASMODIOPHORA SPP.) - Google Patents

Abstract

The present invention provides isolated probes and primers for the detection of Plasmodiophora spp., in particular P. brassicae in plant tissues, and methods therefor.

Description

METHOD OF DETECTING Plasmodiophora spp.

FIELD OF THE INVENTION

The present invention relates generally to novel marker sequences isolated of a soil- borne fungi which infects plants and the uses of said sequences as diagnostic agents for the detection of said fungi in a test sample. In particular, the present invention provides marker sequences the clubroot agent Plasmodiophora brassicae and synthetic oligonucleotide derivatives, homologues, analogues and fragments thereof. The marker sequences of the present invention are particularly useful in the diagnosis of plants which are infected by fungi such as Plasmodiophora brassicae and relatives thereof. The invention provides further, a novel, reliable diagnostic assay for the detection of Plasmodiophora brassicae and relatives thereof in plants, in particular cruciferous plants.

GENERAL

Bibliographic details of the publications referred to in this specification are collected at the end of the description.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular specified source or species, albeit not necessarily directly from that specified source or species.

Throughout this specification and in the claims that follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of steps or elements or integers.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Sequence identity numbers (SEQ ID NOS.) containing nucleotide and amino acid sequence information included in this specification are collected after the Abstract and have been prepared using the programme Patentln Version 2.0. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1 , <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1 , <400>2, etc).

BACKGROUND TO THE INVENTION

Clubroot of crucifers is a severe disease without an effective method for control which causes economic losses and restricts cropping worldwide, including Victoria, Australia (Department of Agriculture, Victoria, Australia, 1983). The disease affects all plants in the crucifer family Brassicaceae, which includes such economically important crops as canola, cabbage, cauliflower, brussel sprouts, turnip, swede, mustard, kohl rabi, kales and buk choy. Affected roots swell markedly into club- and spindle-shaped tumour-like growths, restricting water and mineral uptake by the plants and rendering the roots liable to infection by other pathogenic soil organisms. Affected plants become stunted, yellow and unmarketable, causing economic loss to the growers. Worse, the disease is soil-borne and affected areas become unfit for crucifer production, leading to loss of land value as well.

The disease is caused by a soil-borne fungus, Plasmodiophora brassicae, a fungus which is an obligate pathogen of crucifers. The disease cycle starts with walled resting spores which persist indefinitely in the soil. They germinate in the presence of crucifer roots to produce motile zoospores which attach to root hairs and inject the spore body as a plasmodium, a wall-less growth. The fungus reproduces asexually by the plasmodium cleaving into secondary zoospores, which are released outside the root. These fuse in pairs and reinvade the root to produce plasmodia in which the nuclei are paired. These plasmodia spread to adjacent cells and cleave into many separate multinucleate plasmodia. The presence of the plasmodia causes the plant cells to swell and produce abnormal metabolites. The paired nuclei eventually fuse and resting spores are produced, to be released into the soil when the root disintegrates. The pairing and fusion, and some variation in virulence, suggests sexual reproduction.

Control of the disease is poor and economically inviable, mostly because the resting spores are extremely long-lived in the soil. Once an area becomes infected, it is unsuitable for crucifer growth. Such areas exist and are increasing all over the world. Liming reduces spore germination and hence disease, but leads to other problems of mineral availability in the soil. Soil fumigation is prohibitively expensive except in nursery seedbeds, and depends on the use of many non-specific, toxic chemicals likely to be unavailable in future, e.g. chloropicrin, methyl bromide, dazomet. PCNB (pentachloronitrobenzene) at planting helps to mitigate disease effects, but is now unavailable in many countries. Empirical field trials with surfactants and dichlorophen, using mercurous chloride as a standard, only halved the severity (Humpherson-Jones, 1993). Also, control effects may not persist in future, as has been found with most fungicides, and the worldwide trend is to reduce the use of fungicides in horticulture and agriculture.

Host disease resistance has been bred into some varieties of turnip and rutagaba, but persisted only a few years and so was not economically viable (Agios, 1988). Furthermore, it was suspected that the breakdown of resistance was because of the appearance of virulent new races of the pathogen, possibly through sexual means. Also, differences in resistance and virulence are known among host and fungal strains, but not the basis. Resistance in plant hosts has been linked to peroxidase and chitinase activities (Ludwigmuller et al., 1994) and indole-3-acetic acid (Ludwigmuller et al., 1993), but since studies can only be conducted in the presence of the fungus, fungal activities cannot be distinguished separately. No resistance is currently available for the large crucifer crops such as Brassica oleracea (which includes cabbage and cauliflower), although resistance genes have been identified and their inheritance suggests it is partly chromosomal (Voor ps, 1995). Recent attempts to use somatic hybridisation to produce disease-resistant plants of β. oleracea by somatic hybridisation resulted in 23% being resistant (Gerdemannknorck et al., 1994), but the persistence of the resistance and its effectiveness to many strains of the fungus are not yet known. In many other attempts to induce resistance, all F1 hybrids have been fully susceptible, suggesting that resistance is recessive (Voorrips and Visser, 1993), rather than dominant as in kale (Laurens and Thomas, 1993). Because of the variation in the fungus, methods have been developed for working with single-zoospore isolates (Voorrips, 1995), but this causes the different problem of predicting field resistance in the likelihood of sexual variation and hence variation in virulence in the fungus. Without adequate techniques to study the variation in field populations of the fungus, the development of resistance in plant hosts is likely to be empirical at best.

Progress with control of clubroot disease of crucifers is thus limited by four factors: (a) difficulties in detection of the disease in the soil; (b) lack of knowledge of the pathogen life cycle, in particular genetic variation;

(c) lack of knowledge of the basis of host resistance and rapid breakdown in resistance; and

(d) lack of knowledge of the basis of fungicide control and rapid development of resistance in the fungus. Furthermore, known methods are not suitable for preventing the spread of clubroot disease or preventing infestations of crops by Plasmodiophora ssp. because there are no reliable, sensitive methods available for detecting fungal spores in plant support media such as soils.

At present, the only method of testing soil is by a plant-infection test, which takes at least 6 weeks and is too slow when decisions need to be made on purchase or cropping of land, this can and has resulted in unsuspecting buyers being sold land unfit for normal rotation cropping in production horticulture. It is also fairly unreliable, since variation in susceptibility exists among hosts and pathogens (Voorrips, 1995).

The development of rapid and reliable diagnostic assays for the detection of Plasmodiophora in plants is therefore highly desirable.

In work leading up to the invention, the inventors sought to determine the genetic variation which exists between Plasmodiophora ssp. and between isolates of P. brassicae. The information thus obtained provides the means for developing a wide range of diagnostic agents suitable for the detection of P. brassicae in plant tissue and plant support media and for the diagnosis of clubroot in plants.

SUMMARY OF THE INVENTION

The inventors have utilised the low degree of homology between specific genetic sequences of different Plasmodiophora ssp. and between Plasmodiophora ssp. and other soil-borne microorganisms, in particular those which infect plant tissues, to design reliable, genera-specific and species-specific nucleic acid probes/primers and diagnostic assays for the detection of Plasmodiophora. The diagnostic assays described herein provide significant advantages in terms of time-saving, prevention, containment of fungal spread, sensitivity and reliability over currently employed assays based upon the detection of symptoms of clubroot in plants (e.g. plant-infection tests).

Accordingly, one aspect of the invention provides a diagnostic assay for the detection of Plasmodiophora in a test sample derived from a plant or plant support medium, said assay comprising the detection of a P/astnod/op ?ora-specific genetic element or a homologue, analogue or derivative thereof in said test sample.

The present invention is broadly applicable to the detection of any species of Plasmodiophora in a plant or plant support medium, and is not intended to be limited to the specific species exemplified herein.

Means for detecting the P/asmod/op iora-specific genetic element include any nucleic acid based detection system, such as, for example, hybridisation, polymerase chain reaction (PCR), isothermic amplification, and rolling circle amplification (RCA), amongst others, and the invention is not limited in application to the specific format used. In this regard, those skilled in the art will be aware that, once provided with the novel genetic sequences described herein reliable, genera-specific and species- specific diagnostic assays may be easily developed using a variety of known assay formats or nucleic acid detection techniques.

Accordingly, the present invention provides a method of detecting a Plasmodiophora in a biological sample of plants or a plant support medium comprising contacting a probe or primer comprising a nucleotide sequence of a Plasmodiophora spp. rRNA gene with said sample or nucleic acid derived therefrom for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means, wherein said nucleotide sequence is selected from the group consisting of: (i) rRNA gene sequences that are unique to P. brassicae;

(ii) rRNA gene sequences that are highly conserved between P. brassicae and other species of Plasmodiophora; (iii) rRNA gene sequences that are highly variable between different isolates of P. brassicae; and (iv) nucleotide sequences complementary to any one of (i) to (iii). In particular, the present invention provides nucleotide sequences of the ribosomal RNA gene cluster (rDNA) of P. brassicae that are useful in the detection of sub-types of P. brassicae and/or distinguishing P. brassicae from other species belonging to the genus Plasmodiophora.

Figure 2 herein describes a nucleotide sequence of P. brassicae comprising two ITS regions, which can be used for the derivation of additional nucleic acid probes and primers suitable for performing the present invention. Accordingly, the present invention clearly extends to the use of any nucleic acid probe or primer, of at least about 10 nucleotides in length, preferably at least about 15 nucleotides in length, more preferably at least about 20 or even 30 nucleotides in length, derived from the nucleotide sequence set forth in Figure 2 or an ITS region thereof. For the purposes of clarification, the ITS regions of Figure 2 comprise nucleotides in the region of positions 635 to 773 (ITS1), and/or in the region of positions 938 to 1007 (ITS2), of Figure 2.

More particularly, the present inventors have identified nucleotide sequences of the internal transcribed spacer (ITS) region of P. brassicae rDNA, exemplified herein as SEQ ID NOS: 1-4, that, based upon their homologies to the same region in other Plasmodiophora spp., are useful in performing the inventive method.

Accordingly, a particularly preferred embodiment of the invention provides a method of detecting an isolate of P. brassicae in a biological sample of plants or a plant support medium comprising contacting a probe or primer that comprises a nucleotide sequence of the internal spacer transcribed (1ST) region of a P. brassicae rRNA gene or a nucleotide sequence complementary thereto with said sample or nucleic acid derived therefrom for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means. Preferably, the probe or primer according to this embodiment comprises the nucleotide sequence set forth in any one or more of SEQ ID NOs:1-4 or is complementary thereto. The inventive method is sufficiently sensitive to permit the detection of resting spores of Plasmodiophora spp. or genetic material of Plasmodiophora spp. in the starch grains of resting fungal spores.

A further aspect of the invention provides probes and primers suitable for the performance of the inventive method, in particular the nucleotide sequences set forth in any one or more of SEQ ID NOs: 1-4. The invention further extends to complementary sequences to said nucleotide sequences which can also be used in certain hybridisation formats and, as will be known to those skilled in the art, can be readily derived from the exemplified sequences.

Additional probes and primers that are useful in performing the inventive method can be isolated, for example, using the exemplified probes and/or primers described herein. Such means include any known hybridisation or amplification format, Accordingly, the present invention extends further to any isolated rRNA gene of Plasmodiophora or a fragment of said gene isolated by the method of contacting a probe or primer that comprises a nucleotide sequence of the internal spacer transcribed (1ST) region of a P. brassicae rRNA gene or a nucleotide sequence complementary thereto with said sample or nucleic acid derived therefrom for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means. Preferred rRNA gene fragments are those that comprise nucleotide sequences that are unique to P. brassicae, and/or are highly variable between different isolates of P. brassicae, or alternatively, are highly conserved between P. brassicae and other species of Plasmodiophora.

As will be apparent to those skilled in the art, the probes and primers of the invention, and the inventive diagnostic method described herein, are further useful in diagnosing clubroot in plants and plant media, and more particularly, in confirming a diagnosis of clubroot by virtue of providing a clear diagnosis of infection by Plasmodiophora spp., in suspected cases of clubroot. Further aspects of the present invention relate to the provision of specific nucleic acid molecule probes and primers that are useful in performing the inventive method, and diagnostic kits and reagents comprising same.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a copy of a photographic representation showing specific amplification of Plasmodiophora brassicae genetic sequences from different test sample using rDNA- based primers. Lanes are (left to right): top row, 1 Plasmodiophora brassicae, Werhbee resting spores, 2 Plasmodiophora brassicae, Western Australian resting spores, 3 Plasmodiophora brassicae, Lindenow resting spores, 4 starch grains from Lindenow resting spore suspension, 4a as 4, but smaller amount, 5 young leaves, host plant broccoli 'Marathon', 6 young leaves, host plant Chinese cabbage (ECD05), 7 Spongospora subterranea, 8 Rhizoctonia solani, 9 Pythium sp., 10 Streptomyces albus; bottom row, 11 Phytophthora cinnamomi, 12 VerticilHum dahliae, 13 Leptosphaeria maculans, 14 Plasmodiophora brassicae Western Australian resting spores, 15 Penicillium chrysogenum, 16, Aspergillus niger, 17 Fusarium graminearum, 18, 19 bacterial contaminants from resting spore preparations, 20 negative control (no DNA).

Figure 2 is a copy of a schematic representation of the rDNA region amplified from P. brassicae using PCR, said rDNA region comprising partial 18S rDNA, complete ITS1 region, complete 5.8S rDNA and partial ITS2 nucleotide sequences. Numbering indicates nucleotide positions relative to the first nucleotide in the sequence.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the invention provides a diagnostic assay for the detection of Plasmodiophora in a test sample derived from a plant or plant support medium, said assay comprising the detection of a P/asmod/ophora-specific genetic element or a homologue, analogue or derivative thereof in said test sample. In an alternative embodiment, the invention provides a diagnostic assay for the detection of Plasmodiophora in a test sample derived from a plant or plant support medium, said assay comprising the steps of hybridising a Plasmodiophora ssp.- specific genetic element probe or a homologue, analogue or derivative thereof to said test sample and then detecting said hybridisation using a detection means.

According to this aspect, the Plasmodiophora detected using the invention may be any species of Plasmodiophora which carries a nucleotide sequence which is complementary to the Plasmodiophora ssp.-specific genetic element probe.

In a particularly preferred embodiment, the Plasmodiophora being detected is the causative agent of clubroot in cruciferous plants, P. brassicae.

The term "plant support medium" shall be taken in its broadest context to include solid matrices capable of physically supporting a plant, such as soil, vermiculite, agar or equivalent, compost or peat, amongst others, as well as those solid and liquid which are capable of supplying nutrients to support plant growth, for example soil, compost, vermiculite, hydroponic media, tissue culture media, nutrients and watering or feeding solutions, amongst others. Those skilled in the art will be aware that fungal spores, such as those of Plasmodiophora brassicae may be present in any suitable host plant tissue, including decaying plant tissue derived from infected or uninfected live plants or alternatively, the fungus may be present in any plant support medium, largely because of the capability of fungal spores to remain dormant for long periods without a nutrient requirement. Accordingly, the present invention, which is generally applicable to the detection of only 1-10 resting spores in a test sample derived from

1 gram of soil, is not to be limited by the nature of the plant support medium.

The term "test sample" as used herein shall be taken to refer to any organ, tissue, cell, exudate, nucleic acid, protein, nucleoprotein or other material which is derived from an organism such as a plant or alternatively, derived from a plant support medium which is at least capable of containing resting fungal spores of Plasmodiophora ssp. or fungal hyphae or cells or nucleic acid derived therefrom.

By appropriately selecting the test sample, it is possible to utilise the present invention for both the detection of Plasmodiophora in plant tissue or plant support media such as soils, vermiculite, hydroponic media or tissue culture media, amongst others or alternatively, in nutrient solutions and sprays or watering solutions to be applied to plants. Thus, the prior detection of Plasmodiophora ssp. in growth media and nutrient solutions, etc provides a means by which the infection of plant stocks may be prevented or at least minimised. Similarly, early detection of the fungus in plant stocks may assist in the prevention of spread of clubroot throughout crops.

A suitable test sample may be prepared in solution, for example using an extraction buffer or suspension buffer. Such methods are routine to those skilled in the art.

A particular advantage of the present invention is that it may be readily adapted to facilitate the analysis of a test sample derived from any plant tissue, organ or cell and/or any plant support medium. Those skilled in the relevant art will know how to modify the assay of the invention for the purposes of adapting said assay to the analysis of different test samples, where relevant or indicated, without any undue experimentation.

In a particularly preferred embodiment, the test sample may be derived from the root tissue of a plant or alternatively from soil or watering solutions, or cells, nucleic acid molecules and exudates derived therefrom, for example DNA or RNA, amongst others.

The use of crude or purified nucleic acid samples derived from plant roots or plant support media as test samples for the performance of the assays described herein is particularly contemplated by the invention.

The term "P/asmod/op/iora-specific genetic element" shall be taken to refer to any nucleic acid molecule, in particular DNA or RNA, which comprises a part of the complete genetic material of a Plasmodiophora ssp. and which comprises a nucleotide sequence which is:

(i) unique to a species of Plasmodiophora in particular P. brassicae; or (ii) highly conserved between different species of Plasmodiophora, but not conserved to a significant degree in other fungi; or (iii) highly variable between different isolates of Plasmodiophora of any species, in particular between different isolates of P. brassicae.

For the purposes of the invention, it is not necessary for the P/asmod/op/iora-specific genetic element to be expressed or be capable of expressing a polypeptide product.

In a preferred embodiment of the invention, the

genetic element is an isolated nucleic acid molecule derived from the ribosomal RNA gene cluster (rDNA) of Plasmodiophora ssp, in particular P. brassicae.

More preferably, the P/asmod/opΛora-specific genetic element is derived from the internal transcribed spacer (ITS) region of Plasmodiophora rDNA, in P. brassicae rDNA. A significant advantage to using rDNA sequences is that they are present at high copy number in the Plasmodiophora genome and thus provide for high sensitivity and species-specific detection of a fungus. In this regard, the inventors have shown that such sequences may be used to detect as little as 1 pg P. brassicae rDNA and more preferably as little as 1 fg P brassicae rDNA in a test sample derived from soil, corresponding to <10 spores per gram of soil.

However, in a particularly preferred embodiment of the invention, the Plasmodiophora- specific genetic element is at least about 60% identical to one or more of the sequences set forth in SEQ ID NOS: 1-4 or a complementary nucleotide sequence, or a homologue, analogue or derivative thereof.

Alternatively, the plasmid or plasmid-like genetic element probe is capable of hybridising under at least low stringency conditions to one or more of the nucleotide sequences set forth in SEQ ID NOs: 1-4 or a complementary nucleotide sequence or a homologue, analogue or derivative thereof.

In a further alternative embodiment, the Plasmodiophora ssp. genetic element probe preferably comprises a sequence of nucleotides of at least about 15 nucleotides, more preferably at least about 18 nucleotides and even more preferably at least about 20 nucleotides of the sequences set forth in SEQ ID NOS: 1-4 or a complement or a homologue, analogue or derivative thereof.

The invention clearly encompasses the use of genetic element probes which are homologues, analogues or derivatives of the sequences set forth in SEQ ID NOs: 1-4, for example probes which at least contain the nucleotide sequences set forth in any one or more of SEQ ID NOs: 1-4 but are longer in length, for example at least about 21-24 nucleotides in length or at least about 25-30 nucleotides in length or longer, by virtue of the inclusion therein of additional nucleotide sequences which do not adversely affect their utility in the instant invention. Homologues, analogues and derivatives particularly contemplated herein are those in which nucleotide sequences are added to the 5'-end of the probes to facilitate integration of the probes themselves amplified DNA into a suitable cloning vector, probes in which there is added a specific reporter molecule or capture molecule to facilitate detection and improved probes containing additional nucleotide sequences to enhance the ability of the base probe to detect Plasmodiophora. In all such cases, the homologue, analogue or derivatives will at least be useful in one or more assay formats described herein. Preferably, such sequences will at least contain sequences at least about 60% identical to one or more of SEQ ID NOs: 1-4 or other internal transcribed spacer region sequence of P. brassicae.

For the purposes of nomenclature, the nucleotide sequences set forth in SEQ ID NOS: 1-4 correspond to 20-mer sequences derived from the internal transcribed spacer (ITS) region of Plasmodiophora brassicae rDNA. The subject sequences are highly specific to P. brassicae and may be used to distinguish P. brassicae genetic material from the genetic material of other Plasmodiophora ssp. or alternatively from other fungi, microorganisms or plant genetic material in the test sample. Thus, the Plasmodiophora-spec\f\c genetic element sequences provided herein as SEQ ID NO: 1-4 are particularly useful by virtue of their ability to specifically detect low quantities of P. brassicae rDNA whilst producing a low frequency of false-negative detections or avoiding such false-negative detections altogether.

For the present purpose, "homologues" shall be taken to refer to an isolated nucleic acid molecule which comprises a nucleotide sequence which is substantially the same as the Plasmodiophora-spec\f\c genetic element described herein or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

"Analogues" of the Plasmodiophora-spec\f\c genetic element set forth herein shall be taken to refer to an isolated nucleic acid molecule which comprises a nucleotide sequence described herein or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in an isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radio nucleotides, reporter molecules such as, but not limited to biotin, dioxygenin (DIG), alkaline phosphatase or horseradish peroxidase, amongst others.

"Derivatives" of a Plasmodiophora-spec\f\c genetic element set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to the sequence of said genetic element or a part thereof. Generally, the nucleotide sequence of the

genetic element may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives include 5' and 3^' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues. Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of the genetic element, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.

The present invention encompasses all such homologues, analogues or derivatives of a

genetic element, subject to the proviso that said homologues, analogues or derivatives are useful in the performance of at least one assay format as described herein. Particularly preferred derivatives include DIG- labelled oligonucleotides, preferably derived from one or more of SEQ ID NOs: 1-4 or a homologue or analogue thereof.

The present invention clearly contemplates diagnostic assays which are capable of both genera-specific or species-specific detection.

Accordingly, in one embodiment, the Plasmodiophora ssp. genetic element probe or homologue, analogue or derivative thereof comprises DNA capable of being used to detect multiple Plasmodiophora ssp.

In particular, the inventive method is useful for distinguishing Plamodiophora ssp. from other fungi, and from bacteria and plants, including Aspergillus ssp., Colletotrichim coccodes, Erisiphe cichoracearum, Fusarium spp., GLiocladium roseum, Leptospheaeria maculans, Penicillium chrysogenum, Phytophthora cinnamoni, Pythium ssp., Rhizoctonia solani, Spongospora subterranea, Verticillium ssp., Bacillus subtilis, Bradyrhizobium ssp., Streptomyces albus, Brassica oleracea, Brassica napus, and at least 15 other unidentified or unclassified bacteria. Accordingly, the present invention is considered useful in distinguishing P. brassicae from any other organism. ln an alternative embodiment, the Plasmodiophora ssp. genetic element probe or homologue, analogue or derivative thereof comprises DNA capable of being used to detect a particular isolates of Plasmodiophora ssp.

Whilst not being bound be any theory or mode of action, the more highly conserved sequences in the genetic element derived from a particular species of Plasmodiophora are particularly useful as genera-specific probes for the detection of any Plasmodiophora, while the less-conserved sequences of said element may be useful as isolate-specific probes for the detection of a sub-group of Plasmodiophora brassicae, for example a sub-group which infects plants within the host species but having a particular genotype placing them within the host range of that isolate as opposed to other isolates, or alternatively which induces a specific form of clubroot in plants. Alternatively, the less-conserved sequences may be used to identify a particular species of fungus.

Furthermore, the diagnostic assay of the present invention may also be adapted to a genera-specific or a species-specific or an isolate-specific assay, by varying the stringency of the hybridisation step. Accordingly, a lower stringency hybridisation may be used to simultaneously detect several different species of Plasmodiophora or alternatively, several isolates of P. brassicae in one or more test samples being assayed, while a high or higher stringency of hybridisation is used to distinguish between different species or isolates.

For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% (w/v) SDS at 28°C. A moderate stringency is defined herein as being a hybridisation and/or wash carried out in 2xSSC buffer, 0.1 % (w/v) SDS at a temperature in the range 45°C to 65°C. A high stringency is defined herein as being a hybridisation and/or wash carried out in O.lxSSC buffer, 0.1 % (w/v) SDS at a temperature of at least 65°C. Those skilled in the art will be aware of equivalent reaction conditions to those described herein for defining the hybridisation stringency. Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. Those skilled in the art will be aware that the conditions for hybridisation and/or wash may vary depending upon the nature of the hybridisation membrane or the type of hybridisation probe used. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of the parameters affecting hybridisation between nucleic acid molecules, reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.

The detection means according to this aspect of the invention may be any nucleic acid- based detection means, for example nucleic acid hybridisation techniques or polymerase chain reaction (PCR). The invention further encompasses the use of different assay formats of said nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others.

Wherein the detection means is an RFLP, nucleic acid derived from the test sample, in particular DNA, is digested with one or more restriction endonuclease enzymes and the digested DNA is subjected to electrophoresis, transferred to a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridised to the P/asmod/op/iora-specific genetic element probe as hereinbefore defined, optionally labelled with a reporter molecule. According to this embodiment, a specific pattern of DNA fragments is hybridised to the Plasmodiophora-spec\f\c genetic element probe, said pattern optionally specific for a particular Plasmodiophora ssp., to enable the user to distinguish between different species of the fungus.

Wherein the detection means is a polymerase chain reaction or a variant of same, one or more nucleic acid primer molecules of at least 12 contiguous nucleotides in length derivable from a

genetic element probe as hereinbefore defined or a homologue, analogue or derivative thereof, is hybridised to its complementary sequence in nucleic acid of the test sample or to nucleic acid derived from the test sample and nucleic acid copies of the sequence in the test sample intervening the complementary sequences is enzymically-amplified.

Those skilled in the art will also be aware that, in one format, the polymerase chain reaction provides for the hybridisation of non-complementary Plasmodiophora genetic element probes to different strands of the template molecule, such that the hybridised probes are positioned to facilitate the 5'→ 3' synthesis of nucleic acid in the intervening region, under the control of a thermostable DNA polymerase enzyme. As a consequence, the polymerase chain reaction provides an advantage over other detection means in so far as the nucleotide sequence in the region between the hybridised Plasmodiophora genetic element probes may be unknown and unrelated to any known nucleotide sequence.

Those skilled in the art will be aware that there must be a sufficiently high percentage nucleotide sequence identity between the Plasmodiophora-speάfic genetic element probe(s) and the sequences in the template molecule to which it(they) hybridise. As stated previously, the hybridisation conditions may be varied to promote hybridisation or to reduce background or non-specific hybridisation.

Preferably, the Plasmodiophora- specific genetic element probe is at least 60% identical to the complement of the nucleotide sequence in the template molecule to which it hybridises. More preferably, the Plasmodiophora-specWic genetic element probe is at least about 80% or even more preferably, substantially the same as the complement of the nucleotide sequence in the template molecule to which it hybridises.

Preferably, the P/as od/^'op/.ora-specific genetic element probe is contained in an aqueous mixture of other nucleic acid primer molecules. More preferably, the nucleic acid primer molecule is in a substantially pure form.

According to this embodiment of the invention, the Plasmodiophora-spec\f\c genetic element probe(s) may comprise inosine, adenine, guanine, thymidine, cytidine or uracil residues or functional analogues or derivatives thereof which are capable of being incorporated into a polynucleotide molecule, provided that the resulting probe is capable of hybridising under at least low stringency conditions to a Plasmodiophora- specific genetic element.

In a particularly preferred embodiment, the P/asmod/^'opΛora-specific genetic element probe comprises the sequence of nucleotides set forth in any one or more of SEQ ID NOs: 1-4 or a complementary strand or a homologue, analogue or derivative thereof.

In a more particularly preferred embodiment, the Plasmodiophora-spec\f\c genetic element probes are hybridised to a Plasmodiophora genetic element contained in the test sample being analysed, as probe pairs, in the combinations comprising SEQ ID NOs: 1-2 or SEQ ID NOs: 3-4 or complementary strands, homologues, analogues or derivatives thereof.

The Plasmodiophora complementary sequence present in the test sample, or a part or fragment thereof which is enzymically-amplified, is defined herein as a "template molecule". The template molecule may be a genetic sequence which is at least 40% identical at the nucleotide sequence level to any one or more of SEQ ID NOs: 1-4 or to its complementary nucleotide sequence.

In a particularly preferred embodiment, the nucleic acid template molecule comprises, in addition to other nucleotide sequences, a sequence of nucleotides derived from or contained within any one or more of the sequences set forth in SEQ ID NOs: 1-4 or a complementary sequence or a homologue, analogue or derivative thereof. ln an alternative embodiment, wherein the detection means is AFLP, the Plasmodiophora-specifc genetic element probes are selected such that, when nucleic acid derived from the test sample, in particular DNA, is amplified, different length amplification products are produced from different Plasmodiophora ssp. The amplification products may be subjected to electrophoresis, transferred to a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridised to the Plasmodiophora genetic element probe as hereinbefore defined, optionally labelled with a reporter molecule. According to this embodiment, a specific pattern of amplified DNA fragments is hybridised to the

genetic element probe, said pattern optionally specific for a particular Plasmodiophora ssp., to enable the user to distinguish between different species of the fungus in much the same way as for RFLP analysis.

The technique of AMD facilitates, not only the detection of a Plasmodiophora in a test sample, but also the determination of nucleotide sequence variants which differ from the Plasmodiophora-spec\f\c genetic element probe used in the assay format.

Wherein the detection means is AMD, the P/asmod/^'op/iora-specific genetic element probe is end-labelled with a suitable reporter molecule and mixed with an excess of the amplified template molecule. The mixtures are subsequently denatured and allowed to renature to form nucleic acid "probe:template hybrid molecules" or "hybrids", such that any nucleotide sequence variation between the probe and the temple molecule to which it is hybridised will disrupt base-pairing in the hybrids. These regions of mismatch are sensitive to specific chemical modification using hydroxylamine (mismatched cytosine residues) or osmium tetroxide (mismatched thymidine residues), allowing subsequent cleavage of the modified site using piperidine. The cleaved nucleic acid may be analysed using denaturing polyacrylamide gel electrophoresis followed by standard nucleic acid hybridisation as described supra to detect the Plasmodiophora nucleotide sequences.

Those skilled in the art will be aware of the means of end-labelling a genetic probe according to the performance of the invention described in this embodiment.

According to this embodiment, the use of a single end-labelled probe allows unequivocal localisation of the sequence variation. The distance between the point(s) of sequence variation and the end-label is represented by the size of the cleavage product.

In an alternative embodiment of AMD, the probe is labelled at both ends with a reporter molecule, to facilitate the simultaneous analysis of both DNA strands.

Wherein the detection means is IRS-PCR, the Plasmodiophora-spec\f\c genetic element probes are selected such that they each include one highly-repetitive restriction enzyme cleavage site, for example Alu\, which is ubiquitous in many genomes. According to this embodiment, the appropriate restriction enzyme cleavage site is selected such that it is ubiquitous in Plasmodiophora genetic element nucleotide sequences. The amplified template DNA is electrophoresed under conditions which facilitate high resolution and optionally probed with identical or different labelled

nucleotide sequences.

Optionally, the amplified template DNA may be end-filled using Klenow fragment of DNA polymerase I or other suitable means, prior to the electrophoresis step.

According to this embodiment, different combinations of probes produce different patterns of amplified template nucleic acid.

Furthermore, with any probe combination used, each Plasmodiophora ssp. will produce a distinctive pattern of amplified template nucleic acid. As a consequence, the detection means is suitable for distinguishing between different Plasmodiophora ssp., in addition to being useful for the detection per se of Plasmodiophora in the test sample. Wherein the detection means is RT-PCR, the nucleic acid sample comprises an RNA molecule which is a transcription product of the Plasmodiophora-specWic genetic element DNA or a homologue, analogue or derivative thereof. As a consequence, this assay format is particularly useful when it is desirable to determine expression of one or more Plasmodiophora genetic element genes.

According to this embodiment, the RNA sample is reverse-transcribed to produce the complementary single-stranded DNA which is subsequently amplified using standard procedures.

The present invention clearly extends to the use of any and all detection means referred to supra for the purposes of diagnosing the presence of Plasmodiophora and/or plant support media in plants. Variations of the embodiments described herein are described in detail by McPherson et al. (1991).

Those skilled in the art are aware that the sensitivity of detection may be increased by performing a large number of amplification cycles using any given primer set. Typically, 10 to 30 amplification cycles may be performed in a single reaction. However, as the number of amplification cycles increases, non-specific amplification products also produced in greater number, leading to higher backgrounds.

In an alternative embodiment of the invention comprises a "nested PCR" format, wherein the product of a first amplification reaction, obtained using a first set of amplification primers, is subjected to subsequent amplification using a second set of amplification primers in which one or two of the primers comprising said first set is non- identical with a primer of said second set.

By "non-identical" is meant not 100% identical and as a consequence, overlapping sequences may be "non-identical" for the purposes of this embodiment.

The benefit of nested PCR is that high sensitivity may be obtained without loss in specificity. In fact, by virtue of the presence of at least one primer in the subsequent amplification reaction which is non-identical to the primers used in the first amplification reaction, the specificity of the reaction is likely to be enhanced. As exemplified herein, the present inventors have shown that a nested PCR approach increases the sensitivity of detection of Plasmodiophora brassicae sequences in soil samples from 1 pg rDNA per gram of soil to 1 fg rDNA per gram of soil, corresponding to an increase in the detection of resting spores from 10 spores per gran of soil to 1 spore per gram of soil.

The

genetic element may be labelled with a suitable reporter molecule. Those skilled in the art will be aware that such possibilities also extend to the primer molecules used in a further amplification reaction which is carried out as part of a nested PCR approach. Additionally, the addition of a suitable reporter molecule using standard techniques and without undue experimentation.

Wherein the detection means is a nucleic acid hybridisation technique, the Plasmodiophora-spec\f\c genetic element probe may be labelled with a reporter molecule capable of producing an identifiable signal (e.g. a radioisotope such as ³²P or ³⁵S or a biotinylated molecule or DIG molecule). Wherein the detection means is a polymerase chain reaction, either the Plasmodiophora-spec\f\c genetic element or the amplification product may be labelled with a reporter molecule.

Alternatively or in addition, a capture probe may be used to detect the reporter molecule or the amplified nucleic acid sequences.

By "capture probe" is meant a molecule which is capable of binding to a Plasmodiophora genetic sequence either directly or via binding to a reporter molecule present in said genetic sequence. A capture probe may itself be labelled with a reporter molecule, particularly when used to bind directly to amplified nucleic acid sequence. By "reporter molecule", as used in the present specification, is meant a molecule which, by its chemical nature, produces an analytically identifiable signal which allows the detection of a specified integer or group of integers. Detection may be either qualitative or quantitative. The most commonly used reporter molecule in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes). In the case of an enzyme assay, an enzyme is conjugated to the capture probe or nucleic acid, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphate, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.

It is also possible to employ fluorogenic substrates, which yield a fluorescent product. Fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to capture probes or nucleic acid without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled capture probe or fluorochrome-labelled Plasmodiophora nucleic acid adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.

Those skilled in the art will be aware that the detection of a reporter molecule in either the amplified DNA or the capture probe bound to the amplified DNA or a different reporter molecule provides for identification of the

genetic element probe and that, following the hybridisation reaction, the detection of the corresponding Plasmodiophora ssp. genetic element in the test sample is facilitated.

In a particularly preferred embodiment, the further amplification reaction incorporates a reporter molecule comprising DIG-labelled deoxynucleotide triphosphates [DIG- dNTPs] to facilitate the production of a derivatized (DIG-labelled) amplification product. Following removal of unincorporated DIG-dNTPs using gel filtration, precipitation, electrophoresis or other art-recognised approaches, the DIG-labelled amplification product may be hybridised to a biotinylated capture probe (Boehringer Mannheim GmbH) to form a coloured complex.

By way of example only, the DIG-labelled amplification product is immobilised onto a solid substrate such as the well of a microtitre plate and the biotin-labelled capture probe is brought into contact with the bound nucleic acid sample. After a suitable period of incubation, for a period of time sufficient to allow formation of a biotin:DIG complex, unreacted material is washed away, and the presence of the complex is determined by development of colour. The solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the DIG-labelled molecule to the insoluble carrier.

Such assay formats are particularly useful for the quantitation as well as the detection perse of Plasmodiophora ssp. in particular P. brassicae in a test sample.

The results may either be qualitative, by simple observation of the visible signal or may be quantitated by comparison with a control sample containing known amounts of DIG. The presence of colour which is greater than two-fold the background density is indicative of the presence of Plasmodiophora brassicae genetic sequences in the test sample at 100-fold greater sensitivity than the nested PCR approach performed in the absence of a reporter molecule. Thus, performance of nested PCR in conjunction with the use of a reporter molecule to detect the presence of the amplification product, further increases the sensitivity of detection to approximately 1-10 resting spores in 100 grams of soil sample.

Those skilled in the art will also recognise that the assay method described herein may be used to identify plants in infected fields which appear to resist infection by Plasmodiophora ssp. in particular P. brassicae and isolates and variates thereof. Such plants are particularly valuable sources of improved germplasm for use in breeding programmes.

Accordingly, the present invention clearly extends to germplasm and plants and plant material which is resistant to or has improved resistance to P. brassicae wherein said plants are identified using the assay method described herein.

A further aspect of the present invention contemplates a kit for convenient detection of a Plasmodiophora ssp. in a test sample.

In an alternative embodiment, the kit of the present invention is also useful for convenient assay of infection by a Plasmodiophora ssp. fungus, wherein the sample being tested is derived from a plant or plant support medium suspected of being infected with said fungus.

The kit of the present invention is compartmentalized to contain in a first compartment, one or more nucleic acid molecules which comprise a sequence of nucleotides corresponding to a Plasmodiophora-spec\f\c genetic element or a complementary nucleotide sequence or a homologue, analogue or derivative thereof as hereinbefore defined.

In a particularly preferred embodiment, the first compartment is adapted to contain one or more nucleic acid molecules which are at least about 60% identical to the nucleotide sequence set forth in any one or more of SEQ ID NOs: 1-4 or a complement or a derivative, homologue or analogue thereof.

The kit optionally comprises several second containers comprising a reaction buffer suitable for use in one or more of the detection means described herein and optionally several third containers comprising a nucleic acid molecule positive standard, to which the assay sample result may be compared.

In an exemplified use of the subject kit, a negative control reaction is carried out in which the contents of the first container are contacted with the contents of the second container. At the same time, the sample to be tested is contacted with the contents of the first and second containers for a time and under conditions sufficient for hybridisation to occur. If the reagents contained in the first container provided are not labelled with a reporter molecule, then the contents of the first container may be so labelled prior to the hybridisation reaction being carried out. The hybridised test sample and the negative control sample are then subjected to a detecting means as hereinbefore described. In analysing the results obtained using said kit, the control negative control reaction, test sample and nucleic acid molecule positive standard are compared side-by-side. The contents of the third container should always provide a positive result upon which to compare the results obtained for the negative control and test sample. If the results of the test sample are identical to the results obtained for the negative control, then the test sample does not contain Plasmodiophora ssp. at detectable levels. However, if the test sample produces a nucleic acid molecule which is similar or the same as that contained in the positive standard, albeit of different intensity, then the test sample contains Plasmodiophora ssp.

A further aspect of the invention provides a genera-specific or species-specific or isolate-specific marker, derived from Plasmodiophora ssp.

As used herein, the term "marker" refers to any isolated nucleic acid molecule capable of being used as a genetic element probe according to any of the embodiments described herein to identify a Plasmodiophora ssp., in particular P. brassicae in a plant or plant support medium or alternatively which is capable of being used as a genetic element probe according to any of the embodiments described herein to identify a plant which is resistant to a particular isolate, strain or species of Plasmodiophora or germplasm or plant material derived therefrom. In a preferred embodiment, the marker is derived from Plasmodiophora brassicae or a variant thereof. More preferably, the genetic element probe is derived from the rDNA and even more preferably from the ITS region of rDNA of P. brassicae.

In a particularly preferred embodiment, the genetic element probe comprises a sequence of nucleotides which is at least about 60% identical to the sequence set forth in any one or more of SEQ ID NOs: 1-4, or a complementary nucleotide sequence, homologue, analogue or derivative thereof.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1 Plasmodiophora brassicae samples Clubroot samples were collected from several sites in Victoria (e.g. Werribee, Gippsland) and Western Australia, to give as wide as possible a range of diversity within these areas.

Soil was collected within an area to be tested by taking ten samples per hectare randomly placed within the area. At each point, approximately one trowel-full of topsoil was collected and placed inside a polyethylene bag. The soil samples were mixed together and then three subsamples of 100 mg taken for testing for each hectare.

Water was collected from a dam, watercourse or reticulated supply by taking ten 100 mL samples of water and basal mud. The water samples were mixed together and concentrated by microfiltration to 1 mL and then three subsamples of 100 μL taken for testing.

Plant material (debris) was collected as required, mixed together, ground finely and then three subsamples of 100 mg taken for testing. EXAMPLE 2 DNA Methods

1. DNA Primers The DNA sequence of the ITS region of the rRNA genes (rDNA) was obtained from Genebank using ANGIS databases and aligned.

Sequences specific for P. brassicae were identified and specific primers (forward and reverse) designed and synthesized (SEQ ID NOs: 1-4).

2. DNA extraction from resting spores

The resting spores in the swollen roots ('galls') were extracted and purified by maceration, differential centrifugation through a density gradient of Ludox (Du Pont). This separated the spores from large fragments such as plant cell walls, but starch grains and bacterial contaminants sedimented with the spores, a problem which was partially resolved by adjustment of the Ludox gradient. DNA extraction was attempted using previously published methods (Buhariwalla et al., 1995b) involving digestion of the spore wall using proteases, e.g. proteinase K, and chitinase, since the spore wall is 34% protein, 25% chitin and >17.5% lipid (Moxham and Buczacki, 1983). After the published digestion time, the aim was to embed the resulting spheroplasts in low melting-point agarose for subsequent DNA extraction for PCR according to the published protocol. This proved unsuccessful, in that little DNA was extracted despite extending the digestion times and trying different enzyme brands and batches.

The spores were examined with the scanning electron microscope after freeze-drying to examine the extent of the digestion achieved. Only 5-10% of the enzymatically treated spores were affected, in that the cell wall was sufficiently digested to allow the spore to lose its round shape. The remaining 90-95% resembled the untreated spores in that the cell wall appeared to be unaffected. This method also had the disadvantage that it was taking up to a week to attempt to digest the spore wall, expensive enzymes were required and it was not appropriate to the aim of developing a rapid, simple method for diagnostic use. Other methods were therefore attempted for DNA extraction.

The CTAB (cetyltrimethylammonium bromide) (Richards et al., 1994) method yielded no DNA from resting spores, but gave large quantities of DNA from root galls. However, this would be heavily contaminated by plant host DNA which could interfere with PCR, even using specific primers. A mechanical disruption of the resting spores in NaOH yielded small quantities of DNA, sufficient for PCR.

3. DNA extraction from soil and/or plant material

DNA was extracted by grinding 100 mg of soil or plant material for 2 min in an Eppendorf tube with several grains of acid-washed sand in 300 μL 0.3 M NaOH and 10 μL β-mercaptoethanol. Water is treated similarly but no sand or grinding is needed. An equal volume of phenol hloroform (1 :1 ) is added and the Eppendorf tube is centrifuged at 14,000 rpm (20,000 g) for 5 min, after which the upper aqueous phase is removed and transferred to a new Eppendorf tube. Nucleic acids are precipitated with 2.5 volumes (1000 μL) of ice-cold absolute ethanol and the tube is kept on ice for 10 min and centrifuged at 14,000 rpm (20,000 g) for 10 min. The supernatant is discarded and the pellet resuspended in 200 μL of TE (10 mM Tris(hydroxymethyl)aminomethane, 1 mM EDTA, pH 7.4). RNA is digested by treatment with 1 μL of 10 mg mL^"1 RNase A.

4. Amplification Reaction conditions

1. A volume of 1 μL of DNA extract is put into a PCR (polymerase chain reaction) of the following components:

1 μL DNA extract

0.25 U Tth DNA polymerase

1 mM MgCI₂

60 ng forward primer PblTSI (SEQ ID NO: 1) 60 ng reverse primer PblTS2 (SEQ ID NO: 2)

Buffer as supplied with Tth DNA polymerase Sterile distilled water to make a total volume of 25 μL per reaction

2. The reaction volume of 25 μL is overlain with 50 μL of sterile paraffin oil during the reaction to avoid loss of liquid during the reaction.

3. The first round PCR conditions are as follows:

4. The second PCR uses 1 μL of the product of this first reaction as DNA template in a reaction with the following components:

1 μL DNA template from the fist PCR reaction

0.25 U Tth DNA polymerase

1 mM MgCI₂

60 ng forward primer PblTS3 (SEQ ID NO: 3) 60 ng reverse primer PblTS4 (SEQ ID NO: 4)

Buffer as supplied with Tth DNA polymerase

Sterile distilled water to make a total volume of 25 μL per reaction

5. The reaction volume of 25 μL is overlain with 50 μL of sterile paraffin oil during the reaction to avoid loss of liquid during the reaction.

6. The second PCR conditions are as follows:

Products are visualised using u/v illumination following electrophoresis on 1.5% (w/v) agarose gels containing 0.2% (w/v) ethidium bromide.

5. Results

The amplification reactions used, in particular the nested PCR gave a single band on PCR and agarose gel electrophoresis with resting spore DNA and root galls (Figure 1 ). The results suggest that the PCR primers specifically amplify only P. brassicae DNA, with no amplification from the closely related Spongospora subterranea or host plants. Further testing is being performed, especially of other piasmodiophorids. Also, P. brassicae isolates from around Victoria and Western Australia were tested, and all gave amplification products (Figure 1).

Due to the high copy number of rDNA, high sensitivity can be achieved. In this case, a single set of primers gave a detectable amplification product with 1 pg of template DNA. This level is improved to 1 fg when the nested primers are used, which translates to <10 spores per gram of soil.

The method is currently being adapted to detect viable resting spores only be development of a method which stimulates germination of primary zoospores in soil. The zoospores can then be used in subsequent DNA extractions and PCR amplifications, giving an indication of live P. brassicae spores. If this can be linked to a quantitative PCR, then the assay will be a valuable tool for determining the suitability of soils for crucifer growing. EXAMPLE 3 Determination of the ITS sequence from P. brassicae strains

Amplification products comprising rRNA gene sequences were obtained using first- round primers and the nucleotide sequence of the amplified DNA fragment was determined using standard procedures (Figure 2).

The nucleotide sequence set forth in Figure 2 comprises 1007 bp in length, wherein nucleotides 1 to 634 comprise partial 18S rRNA gene sequence, nucleotides 635 to 773 comprise the ITS1 region, nucleotides 774 to 937 comprise the 5.8S rRNA gene, and nucleotides 938 to 1007 comprise the partial ITS2 region.

The reverse nested primer of the invention hybridises within the ITS1 region, whilst the forward nested primer of the invention comprises a nucleotide sequence within the 18S rRNA gene indicated in Figure 2.

EQUIVALENTS

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. REFERENCES

1. Agrios, G.N. (1988). Plant Pathology, 3rd edn. Academic Press, San Diego, Ca, USA. pp. 268-291.

2. Bock, M., Maiwald, M., Kappe, R., Nickel, P. and Naher, H. (1994). Polymerase chain reaction-based detection of dermatophyte DNA with a fungus-specific primer system. Mycses 37, 79-84.

3. Buhariwalla, H. and Mithen, R. (1995). Cloning of a Brassica repetitive DNA element from resting spores of Plasmodiophora brassicae. Physiological and Molecular Plant Pathology 47, 95-101. 4. Buhariwalla, H., Greaves, S., Magrath, R. and Mithen, R. (1995). Development of specific PCR primers for the amplification of polymorphic DNA from the obligate root pathogen Plasmodiophora brassicae. Physiological and Molecular Plant Pathology 47 , 83-94.

5. Castlebury, L.A., Maddox, J.V., Glawe, D.A. (1994). A technique for the extraction and purification of viable Plasmodiophora brassicae resting spores from host root tissue. Mycologia 86, 458-460.

6. Farbey, M., Dobrowolski, M., Nicholson, C, and O'Brien, P.A. (1995). Detection of fungal pathogens by polymerase chain reaction. 10th Biennial Australasian Plant Pathology Society Conference, Lincoln University, New Zealand, 28-30 August 1995. Abstract No. 246.

7. Dept of Agriculture (1983). Lists of diseases recorded on fruit and vegetable crops in Victoria before 30 June, 1980. Technical Report Series No. 66, Department of Agriculture. Government of Victoria.

8. Figdore, S.S., Ferreira, M.E., Slocum, M.K., Williams, P.H. (1993). Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L. Euphytica 69, 33-44.

9. Gerdemannknorck, M., Sacristan, M.D., Braatz, C, Schieder, O. (1994). Utilization of asymmetric somatic hybridization for the transfer of disease resistance from Brassica nigra to Brassica napus. Plant Breeding - Zeitschrift fur Pflanzenzuchtung 113, 106-113.

10. Humpherson-Jones, F.M. (1993). Effect of surfactants and fungicides on clubroot (Plasmodiophora brassicae) of brassicas. Annals of Applied Biology 122, 457-465.

11. Laurens, F. and Thomas, G. (1993). Inheritance of resistance to clubroot (Plasmodiophora brassicae Wor.) in kale (Brassica oleracea ssp. acephala L.).

5 Hereditas 119, 253-262.

12. Ludweigmuller, J., Bendel, U., Thermann, P., Ruppel, M., Epstein, E., Hilgenberg, W. (1993). Concentrations of indole-3-acetic acid in plants of tolerant and susceptible varieties of Chinese cabbage infected with Plasmodiophora brassicae Woron. New Phytologist 125, 763-769.

10 13. Ludwigmuller, J., Thermann, P., Pieper, K. and Hilgenberg, W. (1994). Peroxidase and chitinase isoenzyme activities during root infection of Chinese cabbage with Plasmodiophora brassicae. Physiologia Plantarum 90, 661-670.

14. Moxham, S.E. and Buczacki, S.T. (1983). Chemical composition of the resting spore wall of Plasmodiophora brassicae. Transactions of the British

15 Mycological Society 80, 297-304.

15. Richards, E., Reichardt, M. and Rogers, S. (1994). Preparation of genomic DNA from plant tissue. In Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (Eds). Current Protocols in Molecular Biology, Vol. 1 , Unit 2.3, Supplement 27.

20 16. Voorrips, R.E. (1995). Plasmodiophora brassicae - aspects of pathogenesis and resistance in Brassica oleracea. Euphytica 83, 139-146. 17. Voorrips, R.E. and Visser, D.L. (1993). Examination of resistance to clubroot in accessions of Brassica oleracea using a glasshouse seedling test. Netherlands Journal of Plant Pathology 99, 269-276.

25 18. Wang, H., Qi, M. and Cutler, J. (1993). A simple method of preparing plant samples for PCR. Nucleic Acids. Res. 21 , 4153-4154.

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M., Gelfand, D., Sninsky, J. and White, T. (Eds). PCR Protocols. Academic

30 Press, San Diego, USA

Claims

CLAIMS:

1. A method of detecting a Plasmodiophora in a biological sample of plants or a plant support medium comprising contacting a probe or primer comprising a nucleotide sequence of a Plasmodiophora spp. rRNA gene with said sample or nucleic acid derived therefrom for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means, wherein said nucleotide sequence is selected from the group consisting of:

(i) rRNA gene sequences that are unique to P. brassicae;

(ii) rRNA gene sequences that are highly conserved between P. brassicae and other species of Plasmodiophora; and (iii) nucleotide sequences complementary to (i) and/or (ii).

2. The method of claim 1 wherein the rRNA gene sequence is a P. brassicae rRNA gene sequence.

3. The method of claim 1 or 2 wherein the rRNA gene sequence comprises internal spacer transcribed region rRNA sequence.

4. The method according to claim 3 wherein the probe or primer comprises a nucleotide sequence that is at least 60% identical to any one of the nucleotide sequences set forth in any one of SEQ IDS NOs: 1 to 4 or a complementary sequence thereto.

5. The method according to claim 4 wherein the probe or primer comprises a nucleotide sequence set forth in any one of SEQ IDS NOs: 1 to 4 or a complementary sequence thereto.

6. The method according to any one of claims 1 to 5 wherein the hybridisation step is performed under low stringency hybridisation conditions.

7. The method according to any one of claims 1 to 5 wherein the hybridisation step is performed under moderate stringency hybridisation conditions.

8. The method according to any one of claims 1 to 5 wherein the hybridisation step is performed under high stringency hybridisation conditions.

9. The method according to any one of claims 1 to 8 wherein the detection means used to detect the hybridisation comprises identifying a signal produced by a reporter molecule bound to the probe or primer, wherein the reporter molecule is capable of producing an identifiable signal.

10. The method according to claim 9 wherein the reporter molecule is a radioisotope or a non-isotopic reporter molecule such as biotin.

11. The method according to any one of claims 1 to 8 wherein the detection means comprises a polymerase chain reaction (PCR) format using one or more Plasmodiophora rRNA-specific primers or primer pairs.

12. The method according to claim 11 wherein the primer pairs comprise SEQ ID NO:1 and SEQ ID NO:2.

13. The method according to claim 11 wherein the primer pairs comprise SEQ ID NO:3 and SEQ ID NO:4.

14. The method according to claim 11 wherein the PCR format comprises a nested PCR.

15. The method according to claim 14 wherein the primer pairs used in the first amplification reaction comprise SEQ ID NO:1 and SEQ ID NO:2, and wherein the primer pairs used in the second amplification reaction comprise SEQ ID NO:3 and SEQ ID NO:4.

16. The method according to any one of claims 1 to 15 wherein the Plasmodiophora detected is an agent of clubroot disease in plants.

17. The method according to any one of claims 1 to 16 wherein the biological sample comprises resting spores.

18. The method according to any one of claims 1 to 16 wherein the biological sample comprises starch grains of resting spores.

19. A method of detecting an isolate of Plasmodiophora brassicae in a biological sample of plants or a plant support medium comprising contacting a probe or primer that comprises a nucleotide sequence of the internal spacer transcribed (1ST) region of a P. brassicae rRNA gene or a nucleotide sequence complementary thereto with said sample or nucleic acid derived therefrom for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means.

20 . The method of claim 19 wherein the probe or primer comprises the nucleotide sequence set forth in any one or more of SEQ ID NOs:1-4 or is complementary thereto.

21. The method according to claims 19 or 20 wherein the hybridisation step is performed under low stringency hybridisation conditions.

22. The method according to claims 19 or 20 wherein the hybridisation step is performed under moderate stringency hybridisation conditions.

23. The method according to claims 19 or 20 wherein the hybridisation step is performed under high stringency hybridisation conditions.

24. The method according to any one of claims 19 to 23 wherein the detection means used to detect the hybridisation comprises identifying a signal produced by a reporter molecule bound to the probe or primer, wherein the reporter molecule is capable of producing an identifiable signal.

25. The method according to claim 24 wherein the reporter molecule is a radioisotope or a non-isotopic reporter molecule such as biotin.

26. The method according to any one of claims 19 to 25 wherein the detection means comprises a polymerase chain reaction (PCR) format using one or more Plasmodiophora rRNA-specific primers or primer pairs selected from the group consisting of: (i) SEQ ID NO:1 and SEQ ID NO:2; and (ii) SEQ ID NO:3 and SEQ ID NO:4.

27. The method according to claim 26 wherein the PCR format comprises a nested PCR, and wherein the primer pairs used in the first amplification reaction comprise SEQ ID NO:1 and SEQ ID NO:2 and the primer pairs used in the second amplification reaction comprise SEQ ID NO:3 and SEQ ID NO:4.

28. The method according to any one of claims 19 to 27 wherein the biological sample comprises resting spores.

29. The method according to any one of claims 19 to 27 wherein the biological sample comprises starch grains of resting spores.

30. An isolated nucleic acid molecule comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1-4.

31. An isolated rRNA gene of Plasmodiophora or a fragment of said gene isolated by the method of contacting a probe or primer that comprises a nucleotide sequence of the internal spacer transcribed (1ST) region of a P. brassicae rRNA gene or a nucleotide sequence complementary thereto with said sample or nucleic acid derived therefrom for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means.

32. The isolated rRNA gene or fragment of claim 31 wherein the probe or primer comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-4 or is complementary thereto.

33. The isolated rRNA gene or fragment of claim 31 or 33 comprising the nucleotide sequence set forth in Figure 2 or an internal transcribed spacer region thereof.

34. Use of a probe or primer comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1-4 to detect Plasmodiophora in a biological sample of plants or a plant support medium.

35. Use of the isolated rRNA gene or fragment according to any one of claims 31 to 30 to detect Plasmodiophora in a biological sample of plants or a plant support medium or to diagnose clubroot in plants.

36. A diagnostic kit for the detection of Plasmodiophora in a biological sample of plants or a plant support medium said kit comprising one or more nucleic acid molecule probes or primers wherein each of said probes or primers comprises a nucleotide sequence of the internal spacer transcribed (1ST) region of a P. brassicae rRNA gene or a nucleotide sequence complementary thereto.

37. The diagnostic kit of claim 36 further comprising a reaction buffer suitable for use in a nucleic acid hybridisation reaction or PCR reaction.

38. The diagnostic kit of claim 36 wherein one or more of the probes or primers comprise a nucleotide sequence set forth in any one of SEQ ID NOs: 1-4 or a complementary nucleotide sequence thereto.

39. The diagnostic kit according to any one of claims 36 to 38 further comprising a Plasmodiophora rRNA gene sequence as nucleic acid molecule positive standard.

40. Use of the method according to any one of claims 1 to 18 in the diagnosis of clubroot in plants.

41. Use of the method according to any one of claims 19 to 29 in the diagnosis of clubroot in plants.