AU677596B2 - Detection of motile organisms in a sample - Google Patents

Description

OPI DATE 26/04/94 APPLN. ID 48117/93 II II lllllll I |ll illllllJIll 11J ill AOJP DATE 14/07/94 PCT NUMBER PCT/AU93/00487lllli il ll 11il AU9348117 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Palent Classification 5 (11) International Publication Number: WO 94/08042 C12Q 1/04, 1/06, COIN 33/569 Al (43) International Publication Date: 14 April 1994 (14.04.94) (21) International Application Number: PCT/AU93/00487 (81) Designated States: AU, CA, JP, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, (22) International Filing Date: 22 September 1993 (22.09.93) NL, PT, SE).

Priority data: Published PL4971 25 September 1992 (25.09.92) AU With international search report.

(71) Applicant (for all designated States except US): THE AUS- v TRALIAN NATIONAL UNIVERSITY [AU/AU]; Acton, ACT 2601 (AU).

(72) Inventors; and Inventors/Applicants (for US only): HARDHAM, A'-ienne, Ruth [AU/AU]; 3/15 Vansittart Crescent, Kambah, ACT 2902 CAHILL, David, Miles [AU/AU]; 31 Carrington Street, Deakin, ACT 2600 (AU).

(74) Agents: SLATTERY, John, Michael et al.; Davies Collison Cave, I Little Collins Street, Melbourne, VIC 3000 (AU).

(54) Title: DETECTION OF MOTILE ORGANISMS IN A SAMPLE (57) Abstract A method for detecting motile organisms, particularly zoospores of Phytophthora cinnamomi in a sample comprises contacting the sample with a solid support in a medium in which the organisms are motile, the solid support being, or being treated with, a chemotactic and/or electrotactic attractant for the organisms, and subsequently detecting organisms attracted to the solid support by im-nunoassay.

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WO 94/08042 PCT/,AU93/00487 -1 "DETECTION OF MOTILE ORGANISMS !N A SAMPLE" FIELD OF THE INVENTION This invention relates to the detection of motile organisms in a sample, and to a device for use in this method. The method is particularly, but not exclusively, directed to the detection of zoospores of the pathogenic fungus Phytophthora cinnamomi. More generally, the method extends to the detection of other organisms that produce motile cells, including bacteria, fungi, algae and protozoa.

BACKGROUND OF THE INVENTION Phytophthora cinnamomi Rands is one of the most important plant pathogens found throughout the tropical and temperate zones (34,36). It affects an increasingly diverse range of species from a wide variety of plant families Hosts include economically important horticultural crops such as avocado, pineapple and macadamia, ornamental species and several valuable timber species. Effects of this fungus on the endemic flora of southeastern Australia and the southwestern part of Western Australia, where some plant species are on the brink of extinction are devastating examples of the impact of an introduced pathogen on a flora composed of many susceptible species (31,34,35). Control of this pathogen and improved understanding of its biology must be based in part on information on the location and density of inoculum in soil. To date, and depending on the method used, this has required relatively difficult and time-consuming procedures to first isolate and then identify P.cinnamomi from soil. It is possible to isolate and identify P.cinnamomi within 2-3 days (13) but many procedures have used baiting of soil samples for up to 7-10 days followed by plating of the infected bait onto one or more selective media. After several days growth on the selective medium it is necessary for identification to be carried out by someone familiar with Phytophthora taxonomy (14,33).

WO 94/08042 PCT/AU93/00487 -2- Currently, the most versatile and useful diagnostic assays are those based on antibodies which specifically r'.cognise the target organism. These assays have been used with great success with plant viral and bacterial diseases (16) and also plant diseases caused by a range of fungi Polyclonal antibodies have been used for the detection of P.cinnamomi.

Antibodies were produced that permitted detection of chlamydospores of P.cinnamomi in soil, but the assay suffered from high background binding of the antibodies to soil particles and lacked specifies-specificity Similar procedures were used to produce antibodies that labelled Phytophthora zoospore cysts and germ tubes, but again these were not species-specific (24).

Several immunoassays were developed for a number of important plant pathogens including Phytophthora For example, monoclonal antibodies were used to detect cysts of Phytophthora and Pythium collected from irrigation water on filter pads These assays all have been based on the use of fungal mycelium or mycelial fractions as the immunogen but have also shown considerable lack of specificity. A collection of monoclonal antibodies raised to aldehyde-fixed zoospores of P.cinnamomi which, in preliminary studies, showed genus-, species- and isolate-specificities have great potential for the development of a species-specific immunodiagnostic test for P.cinnamomi.

It is one object of the present invention to provide a simple, rapid "dipstick" diagnostic immunoassay which in one embodiment, enables the detection and quantitation of P. cinnamomi in soil within 1-2 days. A diagnostic immunoassay of this type is based on an antigen absorbed by, or a capture antibody adsorbed to, a dipstick. These assays involve movement of the dipstick from the test solution through solutions containing a labelled antibody and then into a final solution that allows visualisation of bound antibody. This form of immunoassay has been used successfully to detect several plant pathogens (5,10,11,26) and forms the basis of many medical diagnostic assays (21,32). The great advantages of the dipstick-type assays over other antibodybased assays are that they can be carried out quickly, cheaply and without WO 94/08042 PCT/AU93/00487 -3specialised instrumentation, and a reliable diagnosis can be performed by unskilled workers.

Many organisms are able to detect chemical and electrical gradients, and can actively move towards the source of the gradient, finally adhering to the source. These forms of movement are called chemotaxis and electrotaxis, respectively, and the method of the present invention utilises the chemotactic and/or electrotactic ability of organisms.

It is well documented in the literature that a number of compounds act as chemoattractants for motile bacteria, algal, fungal or animal cells. The present inventors have confirmed and extended studies of chemoattraction of zoospores of Phytophthora species and have determined that the amino acids aspartate, glutamate and asparagine as well as a variety of other compounds are strong attractants of these cells. The inventors have also found that when a solid surface such as a filter or membrane is coated with these compounds, or when the compounds are absorbed by a permeable surface, the slow release of the compounds causes the fungal zoospores to swim up to and adhere to the surface. Materials that have been used successfully include nitrocellulose (Bio-Rad Laboratories), nucleopore filter (Gelman), filter paper Whatman International) and Polysorp and Maxisorp Immunosticks (Nunc, Denmark). In some cases the material was impregnated with chemoattractant alone; in other cases, the material was pretreated with glucose or gelatin.

It is also known that Phytophthora zoospores can detect electrical gradients and exhibit electrotaxis. The present inventors have found that zoospores of P. cinnamomi are strongly attracted to positively charged membranes such as Hybond-N (Amersham Australia Pty Ltd) and zeta-probe nylon membrane (Bio Rad Laboratories). Attraction of the zoospores to these latter membranes occurs in the absence of any applied chemoattractai WO 94/08042 PCT/AU93/00487 -4- SUMMARY OF THE INVENTION According to one aspect of the present invention, there is provided a method for detecting motile organisms in a sample, which comprises contacting said sample with a solid support in a medium in which said organisms are motile, said solid support being an attractant or being treated with an attractant for said organisms; and subsequently detecting organisms attracted to said solid support.

Preferably, the attractant is one which creates a chemical or electric or electro-chemical gradient within the medium. Preferably, also said medium is a liquid medium.

In another aspect, this invention provides a solid support for use in a method as broadly described above, said solid support being an attractant or being treated with an attractant for the organisms. This solid support may be incorporated into a diagnostic or detection kit which further comprises means for detecting organisms attracted to the solid support.

DETAILED DESCRIPTION OF THE INVENTION Central to the method of this invention is the use of a solid support, such as a dipstick, that attracts the organisms of interest. This is preferably achieved in one of two ways. The dipstick may be coated with a compound known to be a chemoattractant for the targeted organism. This compound will diffuse away from the dipstick and establish a gradient of the chemoattractant in the liquid medium. Alternatively or additionally, a support such as a membrane support that possesses an electrical charge may be used to establish an electrical gradient in the liquid medium.

Any suitable method may be used for detecting the targeted organism attracted to the solid support. By way of example, the organism may be detected by use of monoclonal antibodies specific for the targeted organisms.

In the case ofP. cinnamomi, species-specific monoclonal antibodies are known WO 94/08042 PCT/AU93/00487 (17,18), and such antibodies may be used for detection and immunological identification of this fungal species. The monoclonal antibodies are directed towards specific components of the encysted zoospore and preferably recognise antigens on the cyst surface. The assay is specific for P. cinnamomi and shows no cross-reactivity with other Phytophthora species, genera of Oomycetes or plant pathogenic fungi. The sensitivity, specificity and ease of use of the assay will enable widescale application in the forests and crops affected by this pathogen and offers considerable savings in both time and labour.

In general terms, the method of the present invention is based on the application of the phenomenon of chemotaxis and, with the selection of appropriate capture membranes, this can be augmented by electrotaxis to a positive charge. That both of these taxes can be used has implications for the success of the assay. Diffusion of a chemoattractant enables a "long-distance" (several millimetres), short term (minutes), chemical gradient to be established, while the presence of a positive charge in and around the immediate vicinity of the membrane serves as a "close-range", longer term (hours) attractant.

Zoospores of oomycetous fungi are attracted by a variety of chemical compounds including amino acids, sugars, alcohols, aldehydes and phenolic compounds (2,7,19,20,28). Attraction of P.cinnamomi zoospores to several phenolic acids and phytohormones has not been previously reported. In a similar assay, other phenolic compounds, especially the isoflavones, diadzein and genistein, were both potent and specific attractants of zoospores of P.

sojae Of the 65 compounds and mixtures tested in the present study, 32 attracted zoospores of P.cinnamomi at a concentration of 24 mM or less to some degree. The alcohols, amino acids and isovaleraldehyde were the most attractive substances. The present inventors have also demonstrated that the aspartic acid and glutamic acid can induce both attraction and encystment of zoospores of.'.cinnamomi in a manner similar to that found for zoospores of WO 94/08042 PCT/AU93/00487 -6- Py. aphanidermatum Aspartic acid is used as the preferred attractant because it was a potent attractor and induced encystment.

The sensitivity of the dipstick assay of the present invention has been determined to be as few as 40 zoospores mr' in a conical flask. This level of sensitivity is similar to that of an immunoassay for zoospores in irrigation water and detection of ascospores ofVenturia inequalis and greater than that of a chemiluminescence-based assay for bacteria in soil The immunoassay used by the present inventors routinely involved silver enhancement of a colloidal gold-labelled secondary antibody. This detection system produced an intense signal of high contrast that was easily distinguished by eye, hand-held lens or dissecting microscope. The cyst coat material which is immunologically distinct from material which aids in adhesion of cysts spread out around the perimeter of the cyst during assay incubation and hence increased the amount and area of labelling and increased the signal.

In preliminary experiments (data not shown) in which soil was infested with P.cinnamomi the colloidal gold-silver enhancement technique allowed cysts to be readily distinguished from adhering soil and organic particles. The possibility of confusion with other particles adhering to the dipstick membrane, however, was completely eliminated with an alkaline phosphatase-labelled secondary antibody with naphthol phosphate/fast red as the substrate and indicator dye. The pink-red colored cysts were easily distinguished from all other adherent matter. Fast blue, which gives an insoluble light-blue colored product, and bromochloroindoyl phosphate/nitroblue tetrazolium (BCIP/NBT), that gives an insoluble purple reaction product, have also been used to differentiate cysts of P.cinnamomi on dipsticks that have been used in soil samples.

WO 94/08042 PCT/AU93/00487 -7- The length of time used to run the routine assay was 3.5-4 h to maximize labelling and detection in the screening assays, however, for development into a diagnostic kit, assay time could be reduced considerably. In particular, use of alkaline phosphatase conjugated secondary antibody and fast dyes decreased the signal development time to only a few minutes. A biotinstreptavidin-based immunoassay also has the potential to both increase the signal and reduce assay time, With biotin-labelled secondary antibody and streptavidin-labelled with colloidal gold (15 nm), the silver enhancement time was cut to one third that when a gold labelled secondary antibody was used although with increased background signal (data not shown). Process time can also be greatly reduced by making the assay direct rather than indirect by using, for example, a colloidal gold-labelled primary antibody that can then be enhanced. Duration of the procedure was reduced with no loss in sensitivity when this technique was employed with a Cpa primary antibody conjugated to 10-nm gold.

Several immunologically based assays for the detection of plant pathogens are currently available in kit form, and several assays that use monoclonal antibodies as the detection system have been reported (9,27).

Cross-reactivity however, has limited the use of many of these assays, which have immunogens based on whole cells or cell walls and soluble surface antigens. Similarly, the usefulness of commercial diagnostic tests for the detection of Phytophthora and P,.cinnamomi in particular has been severely hampered by cross-reactivity both within the genus and to other closely related genera such as Pythium and Peronospora (1,3,23,29).

Further features of the method and other aspects of the present invention will be apparent from the detailed descriptio in the following Example.

WO 94/08042 PCT/AU93/00487 -8- EXAMPLE 1 MATERIALS AND METHODS Fungal isolates.

The P.cinnamomi isolate (A2 mating type, 6BR, H1000) used throughout this work was originally isolated from the Brisbane Ranges, Victoria, Australia.

Sources and culture conditions for all other isolates of Phytophthora and isolates of Pythium and Saprolegnia have been detailed (13) and have been assigned culture collection identification numbers (Table 1 in Zoospore production.

Zoospores of P.cinnamomi were produced axenically and zoospores of all other fungi were produced with the method of Dolan and Coffey (12).

Numbers of zoospores in suspensions ranged from 1 X I 1 5 X 1 0 ml'.

Monoclonal antibodies.

Monoclonal antibodies (MAbs) from a previously described collection (17,18) were used. The MAbs were grouped according to labelling patterns on zoospores and cysts: Zt binds to the surface of the anterior, tinsel flagellum; Zg binds to a restricted area in the ventral groove and to mastigonemes on the anterior flagellum; Cpa binds to the cyst coat material; Lpv binds to the contents of large peripheral vesicles; Cpw binds to the cyst wall; ZCp binds to the surface of both zoospores and cysts; and Vsv and Gw bind to the contents of ventral surface vesicles located around the groove region.

Chemotaxis assay.

A wide range of analytical-grade chemical compounds, including sugars, amino acids, phenolic compounds, alcohols, organic acids and plant hormones, were tested. The assay for determining the chemoattractiveness of compounds for zoospores of P.cinnamomi was modified from an agar-filled tube technique A chemotaxis chamber was formed by attaching two 5-gl microcapillary tubes longitudinally, 13 mm apart, in the center o: a glass microscope slide WO 94/08042 PCT/AU93/00487 -9- (25.4 X 72.6 mm) and then placing a glass coverslip (22 X 22 mm) over the capillary tubes to form an open-ended chamber (240 p in volume). Zoospore suspension was carefully introduced into the chamber from a glass pipette. The test substance, dissolved in distilled water, was drawn into a 5-ipl capillary tube and inserted 7 mm into one end of the chamber. A tube that contained distilled water alone was inserted into the opposite end of the chamber and served as the control in each test. Four slides were prepared for each test substance.

Slides were immediately transferred to a humidity chamber that was then kept in the light at room temperature (approximately 25 After incubation for min, slides were removed from the chamber and heated for 5 sec on a hotplate to immobilize the zoospores. Counts of zoospores within the capillary tubes were made from images produced from a videographic printer (Sony (Australia) Pty. Ltd., North Ryde, NSW, 2113) coupled to a monitor and video camera mounted on a Zeiss Photoscope III microscope. Zoospores were counted individually by an electronic colony counter (Manostat, New York, NY, USA).

Mean number of zoospores in the test and control capillaries were calculated, and depending on the ratio of the number of zoospores in the capillary containing the attractant to the number of zoospores in the capillary without attractant the test compounds were rated as not attractive (ratio of weakly attractive moderately attractive or strongly attractive >2.5)(Table Tests were performed at least twice on each test substance.

Dipstick design.

In preliminary experiments, there was used a range of solid supports on which zoospores were able to encyst and adhere. Supports included plastic, glass, filter paper, an Immunostick (Nunc, Denmark), nitrocellulose membranes (plain nitrocellulose and supported nitrocellulose from Bio-Rad, North Ryde, NSW, 2113 and Hybond- C and Hybond Super-C from Amersham Australia Pty Ltd, North Ryde, NSW, 2113), nylon membranes (zeta-probe and zeta-probe GT, both positively charged, from Bio-Rad, and Hybond-N and Hybond-N the latter positively charged, from Amersham), and a polyvinylidene difluoride (PVDF) membrane from Bio-Rad. These supports were variously treated for use WO 94/08042 PCT/AU93/00487 in several assay types (Table In some assays, an attractant was added to the membrane square which, in several tests, was attached to one end of a plastic support by clear nail varnish. In other assays, the attractant was added to filter paper strips or mixed with gelatin, poly-L-lysine or glucose and applied to the solid support. The effectiveness of the dipstick format in attracting and capturing the zoospores was assessed by counting the number of cysts that had adhered to the dipsticks after a 30-min incubation in a zoospore suspension (104 mr 1 Dipsticks that did not contain attractant served as controls.

Dipstick immunoassay.

Several direct and indirect immunoassays were assessed in preliminary experiments. In initial tests, the prese'rce of cysts on the dipstick was tested with an alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibody enzyme reaction to produce a colored solution, but these assays were successful only when high numbers of cysts (500 1000) were attached to the membrane (data not shown). Methods based on the formation of an insoluble, colored precipitate on and around the cysts were far superior. An indirect immunoassay with a colloidal gold (10 nm)-labelled secondary antibody (AuroProbeTM BL plus GAM IgG IgM (H Amersham) and subsequent silver enhancement (IntenSE TM BL silver enhancement kit, Amersham) was used routinely in all subsequent experiments. During development of the method, assays were run in 96-well ELISA trays and the total assay time was 3.5 4 h.

The time taken for the assay was reduced to less than 45 min without loss of sensitivity by reducing incubation times and by increasing the concentrations of the primary and secondary antibodies.

For the routine assay a dipstick was placed in 200 il of zoospore suspension for 30 min and transferred to 250 ~l of 5% skim milk (Carnation, Sydney, NSW, 2000) in Tris-buffered saline, (TBS) pH 7.4, with 0.1% gelatin for min. The dipstick was washed twice for 3 min in 250 pi TBS containing 0.8% bovine serum albumin (Sigma Chemical Co., Castle Hill, NSW, 2154) WO 94/08042 PCT/AU93/00487 -11 (TBS/BSA) and placed int, 200 p1 of genus- or species-specific monoclonal antibody appropriately diluted in TBS/BSA. After incubation for 45 min, the dipstick was washed twice for 3 min in 250 p TBS/BSA and placed into 200 p1 of gold-labelled goat anti-mouse IgG IgM (H L) for 45 min. The dipstick was then washed once in 250 pl TBS/BSA for 3 min, once in 250 il TBS for 3 min, and in 250 pC of distilled water for 1 min. The dipstick then was transferred to 250 pl of silver enhancement reagent. Within 60-70 min, an intense black precipitate formed on and around cysts, which had bound the primary antibody.

The reaction was stopped by immersing the dipstick in distilled water for 1-2 min. The dipstick was then air-dried, and cysts were observed unmagnified or with a 10X hand lens. Dipsticks were critically examined with a stereo dissecting microscope with zoom optics (maximum magnification 67.5X) and incident illumination. All steps in the protocol were conducted in a laboratory at room temperature under constant lighting conditions. In assays for which counts of the total number of cysts on each dipstick were required, the procedure used for counting cysts in the chemotaxis chambers was followed. Dipsticks were attached to microscope slides with adhesive tape and illuminated from above with a dissecting microscope light source.

Application of the chemoattractant.

Aspartic acid or glutamic acid were dissolved in distilled water at 95 C.

A 1-pl aliquot of a suitably diluted solution of the amino acid was placed in the center of the membrane square. The dipsticks were then use immediately or stored desiccated. To test the effectiveness of these two compounds after application to the dipstick membrane, dipsticks were incubated in a zoospore suspension for 30 min, and cysts were counted as described above.

Monoclonal antibody screening.

Twenty-four MAbs, selected from the immunofluorescence assay as potentially diagnostic from an original collection of 35 MAbs were screened for the ability to bind to cysts in the dipstick assay. Affinity purified antibodies (1 or 10 pg mr 1 or hybridoma supernatants (neat or 50% dilution in WO 94/08042 PCT/AU93/00487 -12- TBS/BSA) were used in the indirect immunoassay and each was scored after silver enhancement of the secondary probe relative to a non-immune mouse IgG (NIM) negative control (10 ig mr') for their ability to bind to cysts that were attached to the dipstick membrane and to produce a visible precipitate. MAbs were tested on duplicate dipsticks with P.cinnamomi (6BR, H1000) cysts.

Cross-reactivity of Phytoxphtra and related genera.

Cross-reactivity of the MAbs with cysts of isolates of Phytophthora Pythium or Saprolegnia was tested with the dipstick immunoassay. The Cpa MAbs (Cpa-2, 1 vg m'r of the purified MAb; Cpa-3, 25% dilution of hybridoma supernatant and Cpa-7, 50% dilution of hybridoma supernatant) and ZCp-2 dilution of hybridoma supernatant) and Cpw-4, (10 |g mr 1 of purified MAb) were tested. NIM (10 g mr 1 was used as the negative control for each isolate. All MAbs were diluted in TBS/BSA. MAb Vsv-l, which recognises an antigen within the ventral surface vesicles of zoospores in each of the genera, was used as a check for the presence of cysts of Pythium and Saprolegnia on the dipstick membrane since cysts of these genera were not labelled by the MAbs tested. The dipsticks were joined in strips of 12 so that they could be used in a 96-well ELISA plate assay.

Sensitivity of the dipstick assay.

Three procedures were compared and used to determine the dipstick assay sensitivity. A dilution series of zoospores from an initial concentration of zoospores mr' was used for each. In the first procedure, the zoospore suspension was diluted with distilled water to a range of concentrations in a total volume of 12 ml and was added to a 10 ml Erlenmeyer flask so that the surface of the zoospore suspension was approximately half-way up the neck (12 mm internal diameter) of the flask. In the second procedure, 10 ml of zoospore suspension was dispensed into small glass Petri dishes (40 mm diameter). A single dipstick was placed, membrane side down, on the surface of the zoospore suspension in each flask or Petri dish. In a third procedure, dipsticks were used in an ELISA tray assay in which zoospores were diluted WO 94/08042 PCT/AU93/00487 -13with distilled water to form a dilution series. Dipsticks were placed vertically into 200 pil of zoospore suspension within the well of the tray. After incubation for min in the zoospore suspensions, dipsticks were removed and subjected to immunoassay. Cysts on the dipstick membrane were counted as described above.

RESULTS

The results are set out in Tables 1-4 as follows: Table 1: Relative attractiveness of compounds for zoospores of F.cinnamomi determined using an assay in which the cells swim into a capillary from which the test compound is diffusing.

Table 2: The effectiveness of various solid supports, assays, coatings and chemoattractants for the capture and assay of zoospores of P.cinnamomi.

Table 3: Comparison of amino acids (1mM) in the chemotaxis assay for attractiveness to P.cinnamom! zoospores.

Table 4: Screening results obtained in the dipstick assay to determine the reaction of 24 monoclonal antibodies with differing binding characteristics for labelling of cysts of P.cinnamomi Selection of chemoattractant(s).

Compounds that were chemotactically attractive to zoospores of P.cinnamomi were identified in the "swim-in" assay (Table Many of the compounds tested caused rapid accumulation of zoospores within the capillary tubes. The compounds included those known to occur in the rhizosphere, root exudates, and roots of many plant species e.g. Of the compounds tested, several amino acids, alcohols, phenolic compounds and WO 94/08042 PCT/AU93/00487 -14isovaleraldehyde were highly attractive. The amino acids aspartic acid and glutamic acid, were especially attractive (Table Some compounds, including pectin, syringic acid, abscisic acid, aspartic acid and glutamic acid, also caused encystment. Sugars were generally not attractive except at relatively high concentrations (>100 mM, data not shown). Preliminary experiments with several of the compounds run in dilution series demonstrated that there was a concentration for each compound (usually >100 mM) above which zoospores were repelled or encysted rapidly and a concentration below which zoospores were not attracted. For example, concentrations of aspartic and glutamic acids that caused the greatest accumulation of zoospores within the capillary tubes differed and was 0.1 mM for aspartic acid or D- configuration) and 1 mM for glutamic acid. On the basis of their attractiveness and encystment-inducing properties, aspartic and glutamic acids, arginine, pectin, and ethanol were chosen for use in development of the dipstick assay.

Dipstick format.

Zoospores were attracted to and encysted upon all the solid supports used (Table The presence of an attractant absorbed into a membrane, mixed with the coating substance or dried to the support surface increased the number of cysts bound to the support compared with controls that did not contain attractant. Cyst numbers were higher on the nitrocellulose and nylon membranes treated with attractant than on ither support types and coatings.

Coated supports such as the immunostick, glass slides and the plastic dipstick attracted zoospores, but the coating material usually did not bind well and was lost after several washes. This made the coatings inappropriate for use in an immunoassay. In contrast, cysts adhered well to the membrane surfaces and were not removed by washing.

The most successful and useful format was the plastic dipstick (5 mm X mm) with a square (5 mm X 5 mm) of nitroce!lulose or nylon membrane glued to one end. A variety of membrane types were tried but the nylon membranes, which are more hydrophilic than nitrocellulose, enabled more even dispersal WO 94/08042 PCT/AU93/00487 and rapid absorption of the water drop containing the dissolved attractant. The PVDF membrane, although used successfully by others was unsuitable because it must be wetted with methanol before use. Positively charged nylon membranes attracted zoospores in greater numbers with or without added chemoattractant than neutral membrane. The zeta-probe nylon membrane, which carries a high-density quaternary amine charge, was especially attractive.

These plastic dipsticks with attached membrane had several advantages over the other supports and coatings. They could be used vertically in or horizontally on a zoospore suspension and could be used in 96-well ELISA trays either as single sticks or, when correctly aligned on a length of adhesive tape, in strips of up to twelve. The use of multiple dipstick strips enabled the processing of many samples simultaneously and considerably eased the logistics of the immunoassay.

Monoclonal antibody screening.

There was no labelling by MAbs of the Zt group of P.cinnamomi (6BR, H1000) cysts that had adhered to the dipstick membrane (Table There were very small localized areas of labelling with each of the Zg MAbs (possible labelling of the water expulsion vacuole, but these could be seen only under high-power magnification 100X). All the Lpv MAbs gave weak to very weak, diffuse labelling patterns. In contrast, seven of the Cpa MAbs reacted moderately or strongly with material coating the cyst surface. Three of the Cpa Mabs (Cpa-5, Cpa-8 and Cpa-12) showed none or only weak labelling. The Cpw-4 and ZCp-2 MAbs reacted moderately and the Gw MAb only weakly in the assay.

Five MAbs were selected based on the intensity of labelling of cysts in the dipstick assay and on supplementary information from immunofluorescence and ELISA studies MAbs Cpa-2, Cpa-3 and Cpa-7 (putative species-specific) and Cpw-4 and ZCp-2 (putative genus-specific). These MAbs were used in further studies to test for specificity in the dipstick assay.

WO 94/08042 PCT/AU93/00487 -16- Screening Phytophthora and related genera.

Forty-four isolates of P.cinnamomi including 15 Al mating type and 29 A2 mating type, obtained from throughout Australia and including isolates from Papua New Guinea ana Japan, 21 species or varieties of Phytophthora encompassing 75 isolates, 11 species of Pythium encompassing 13 isolates and three species of Saprolegnia (one isolate each) were tested against the five selected MAbs with the dipstick assay. All three Cpa MAbs labelled cysts of the P.cinnamomi isolates but did not label any cysts from isolates of the other Phytophthora species or varieties. In contrast, MAbs Cpw-4 and ZCp-2 labelled all Phytophthora isolates. Slight variation occurred among the P.cinnamomi isolates in labelling intensity of the cysts, but no difference occurred between Al and A2 mating types. The putative species-specific MAbs labelled cysts more strongly than the putative genus-specific MAbs.

MAbs Cpa-3 and Cpa-7 did not label any cysts from the isolates of Pythium and Saprolegnia tested. MAb Cpa-2 did, however, cross-react weakly with Pythium aphanidermatum, Py. butleri, Py. debaryanum, Py. irregulare and S.

declina. MAb ZCp-2 reacted weakly with Py.debaryanum and Py.irregulare.

MAb Cpw-4 reacted weakly with Py.middletonii. MAb Vsv-1, which labels the contents of ventral surface vesicles, reacted with all isolates of Pythium and Saprolegnia.

Dipstick assay sensitivity.

The shape of the container holding the zoospore solution influenced the number of cysts found on the dipstick membrane. Over the range of dilutions of the zoospore suspension more cysts were found on dipsticks used in the conical flask assay than the Petri dish or ELISA plate assays. The sensitivity of the dipstick assay was determined from a dilution series as the highest dilution of zoospores that could be detected. For the conical flask assay, 40 zoospores mf' was the lowest concentration at which one or more cysts were found on the dipstick membrane after the 30 min incubation period. Minimum detection limits were 156 and 312 zoospores m'r for the Petri dish and ELISA plate WO 94/08042 PCT/AU93/00487 17assays, respectively. Results from the ELISA plate assay are, however, not directly comparable withdhose of the other two assays because the dipsticks were placed vertically inthe zoospore solution. A single cyst attached to the dipstick membrane was the minimum required for a positive identification although in practice probably no fewer than 5-10 cysts per membrane would be the minimum necessary to be confident of an identification.

WO 94/08042 WO 9408042PC1'/A U93/00487 18 Compound (molarity) RA Cornpound (molanity I RA ICompound (molarity) IRAI Sugars (10 mM) Amino acids (1 mM) Organic acids (10 mM) D-Marnose L-Asparfic acid Maleic D-Xylose D-Aspartic acid L-Malic D-Ribose L-Metbonine Citric D-Fructose D-Methionine Succinic D-Lactose L-Glutamidc acid Fumaric D-Glucose -L-Arginine L-Ascorbic Sucrose Guanidine Valeric D-Galactose .Glycine Isovaleric L-Fucose L-Cysteine Fofic D-Cellobiose L-Lysine Acetic L-Rhaxnnose .L-Leucine L-Arabinose L-Phenylaanine Pectin and derivatives L-Tyrosine D-Galacturonic acid (100ug/mni) Pheniolics (10 mM) Polygalacruronic acid (1O0ug/mi) Caffeic acid Alcohols (25 mM) Pectin Hydroxybenzoic acid .Ethanol Ferulic acid Methanol -4-i Aldehiyde (1 mM) Syringic acid Isopropanol 4-H- Isovaleraldehyde Gallic acid ('entisic acid Phytohormones (10 M) Miscellaneous p-Counic acid -Kinetin Cascin lOug/ml Vanillin Benzylaminopunine Casein hydrolysate lO0ug/znl Coumnarin Isopentenyladenine Phioroglucinol Abscisic acid V8 juice RutiF, Gibberellic acid (GA3) V8 broth My.;I- Indolbutyric acid Kacmfeol_ Napthalene acetic acid Gelatine RA Relative attractiveness -compound not attractive +,weakly attractive moderately attractive highly attractive cause encystroent WO 94/08042 WO 9408042P CT/A U93/00487 19 Table 2. Effectiveness of various solid supports, assays, coatings and chemoattractants for the capture and assay of zoospores of A~ cinrnonu.

Solid support _Assay format Coating Test compound Ratio Nitrocellulose Square (5 x 5 mm) Nc Aspartic acid membrane floated on zoospore suspension Nc: Arginine Nc Glutamic acid Nc V8 brodfl Nc Pectin Strips (2 x I10mm long) Nc Aspartic acid in chemnotaxis chamber Nc Glutamic acid Nc Ethanol Strips (5 x 10mm long) Nc Aspartic acid in ELISA tray Nc Glutamic acid Nc Pectin Nc Ethanol +4 Immunostick Immersed in zoospore Nc Aspartic acid suspension Nc Glutarnic acid Nc Pectin glucose Aspartic acid glucose Glutarnic acid glucose Pectin Ratio is the number of zoospores on the solid Gupport treated with the attractant divided by number of zoospores on the solid support without attractant where ratio of 1.1 1.5, 1.6 2.5, and 1 i 2.5. Nc not coated.

WO 94/08042 W094/8042PCT/A U93/00487 20 Table 2. (continued) Solid support Immunostick (continued) Assay format Coating 10% gelatin gelatin gelatin Poly-L-Lysine Poly-L-Lysine Poly-L-Lysine 10% gelatin Test compound Aspartic acid Glutamic acid Pectin Aspartic acid Glutamic acid Pectin Glutamic acid Ratio 44+ 44 Plastic dipstick ELISA tray x 20mm long) Poly-L-Lysine Glutamic acid Plastic dipstick x 20mm long) Nitrocellulose membrane (5 x Nylon membrane x Nylon membrane x Strips (2 x 10mm long) in chemotaxis chamber Nc Nc Nc Nc Nc Nc Nc Nc 10% gelatin Aspartic acid Glutamic acid Aspartic acid Glutamnic acid No attractant Aspartic acid Glutaniic acid Ethanol Aspartic acid 44+ +44 +44 44+ 44 44 44 44 Filter paper Glass microscope Immersed in zoospore slide suspension Ralz is the number of zoospores on the solid support treated with the attractantt divided by number of zuospores on the solid support without attractant where ratio of 1. 1 1.5, 1.6 2.5. and i i 2.5. Nc not coated.

WO 94/08042 PCT/AU93/00487 -21 Table 3. Comparison of amino acids (1 mM) in the chemotaxis assay for attractiveness to P. cinnamomi Amino acid Number of zoospores in capillaries Ratio Control amino acid L-Aspartic 24.6 11.2 128.2 32.0 1 D-Aspartic 30.2 7.7 116.8 18.3 1 L-Methionine 26.6 11.8 41.4 15.0 1 1.6 D-Methionine 102.4 10.2# 99.4 21.3# 1 L-Glutamic 20.0 7.3 78.2 18.8 1 3.9* L-Arginine 32.8 6.4 147.4 37.1 1 Mean and standard errror of the mean of four replicates in two separate experiments are shown.

Data are counts from one field of view (X 10 objective) or from two fields of view, Ratio calculated as number of zoospores in the capillaries containing the amino acid divided by the number in capillaries without amino acid.

significantly different from controls, P< 0.1, P< 0.05, P< 0.01.

WO 94/08042 PCT/AU93/00487 22 Table 4. Results of screening 24 MAbs for fkc tiveness of labelling of P. cinnanwmi cysts in the dipstick assay.

MAb Reaction Zt- 1 Zt-2- Zg- I Zg-2 Zg-3 Zg-4 Cpa-2 Cpa-3 Cpa-4 Cpa-6 Cpa-7 Cpa-8 Cpa-9 Cpa- 10 Cpa-12 +1- Lpv-1 Lpv-2 Lpv-3 Lpv-4 Cpw-4 ZCp-2 (3vv -no labelling trace +weak moderate strong WO 94/08042 PCT/AU93/00487 -23- EXAMPLE 2 MATERIALS AND METHODS A. Tests in the glasshouse/constant environment cabinets.

Tests have been carried out in constant environment chambers to determine whether the dipstick assay is effective under glasshouse and field conditions and is specific for P.cinnamomi in the presence of spores of other Phytophthora and Pythium species. Tests have involved the inoculation of several plant species (Eucalyptus sieberi, Pinus radiata, Lycopersicon esculentum and Banksia serrata with isolates of Phytophthora cinnamomiAl, P.citricola, P.nicotianae var nicotianae, P.cryptogea and two Pythium species, Pythium aphanidermatum and Py.irregulare. These isolates have been used singly or in combination with and without the addition of P.cinnamomi A2.

Reisolation of P.cinnamomi A2 has used several different methods. All experiments have used a baiting assay (Eucalyptus sieberi cotyledons/or pine needles) to check and confirm results of the dipstick assay. Baits are plated onto selective media containing antibiotics which inhibit the growth of fungi (other than Phytophthora and Pythium and bacteria and the inoculum present confirmed by traditional taxonomic means. In addition, dipsticks have been plated onto selective media to prove the presence of both spores of P.cinnamomi and the other Phytophthora and Pythium species in the samples taken. In some experiments aliquots of the soil slurry made from the sample taken have been plated onto selective media to again confirm the presence of the isolates of interest.

All the above procedures for reisolating P.cinnamomi work, especially the dipstick and baiting assays. Plating of dipsticks onto selective media has been less reliable (probably because only one dipstick per sample is sampled) for reisolation of P.cinnamomi but has been extremely useful for proving that motile spores of the other species are being produced and are attaching to the membrane on the dipstick. The soil slurry technique, also useful for estimating WO 94/08042 PCT/AU93/00487 -24amounts of fungal material in the sample suffers from contamination by fungi and bacteria which are not inhibited by the selective media.

The dipstick assay has proven extremely reliable for detection of P.cinnamomi under the controlled conditions used. There are only a small number of cases where the dipstick has failed to pick up P.cinnamomi when the baiting assay has been positive. It should be noted that there are also examples of the reverse, i.e. dipstick positive, baiting negative.

B. Tests on soil collected in the field.

The critical test of the dipstick assay is whether it has the ability to detect P.cinnamomi in field soils. Data now exists to show that the assay works in different soil types from several sites around Australia.

The present inventors have used similar isolation and contirmation procedures to those used in the controlled experiments, i.e. plating of baits onto selective media, plating of dipsticks onto selective media and isolation from soil slurries. In addition, identifications from field soil have been rigorously confirmed by isolating putative Phytophthora species, bringing them into pure culture, inducing them to produce zoospores and then running the dipstick assay on the pure zoospore cultures.

The dipstick assay has been completely reliable for identifying P.cinnamomi in the field soils. There have been no cases to date where there is not agreement between baiting and traditional methods of isolation and the dipstick assay.

C. Sensitivity of the dipstick assay.

In the laboratory experiments previously described, it has been determined that the dipatick assay is capable of detecting as few as zoospores per millilitre. In tests where plants grown in soil in pots have been inoculated with a zoospore dilution series (plants used have been Pinus radiata, WO 94/08042 PCT/AU93/00487 Lycopersicon esculentum, and Banksia serrata the dipstick assay and the cotyledon baiting assay are capable of detecting the presence of P.cinnamomi after the addition of 3000 zoospores per pot. In some instances the dipstick assay has detected the presence of P.cinnamomi after the addition of only 300 zoospores per pot.

It will be appreciated that the above Examples are included by way of illustration, and not limitation, of the invention as broadly described herein, and that many modifications and variations may be made without departing from the spirit and scope of the invention.

WO 94/08042 PCI/AU93/00487 -26-

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(1993). Specificities of monoclonal antibodies to Phytophthora cinnamomi in two rapid diagnostic assays. Plant Dis. (In Press).

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Gubler, F. and Hardham, A.R. (1988). Secretion of adhesive material during encystment ofPhytophthora cinnmomi zoospores, characterized by immunogold labelling with monoclonal antibodies to components of peripheral vesicles. J.Cell Sci. 90:225-235.

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18. Hardham, Suzaki, E. and Perkin, J.L. (1986). Monoclonal antibodies to isolate-, species- and genus-specific components on the surface of zoospores and cysts of the fungus Phytophthora cinnamomi.

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Claims (19)

1. A method for detecting motile soil organisms such as Phytophthera cinnamomi in a sample, which comprises contacting said sample with a solid support in a medium in which said organisms are motile, said solid support being an attractant or being treated with an attractant for said organisms wherein said attractant attracts said organisms by chemotaxis and/or electrotaxis; and subsequently detecting organisms attracted to said solid support.

2. A method according to claim 1 wherein said solid support is in the form of a dipstick.

3. A method according to claim 1, wherein organisms attracted to said solid support are detected by immunoassay.

4. A method according to claim 1, wherein said motile organisms are zoospores of Phytophthora cinnamomi and said solid support is treated with an i attractant for said zoospores.

5. A method according to claim 4, wherein said attractant is selected from the group consisting of L-aspartic acid, D-aspartic acid, glutamic acid, arginine, pectin and ethanol.

6. A method according to claim 1 wherein said motile organisms are zoospores of Phytophthora cinnamomi and said solid support comprises an attractant for said zoospores.

7. A method according to claim 6, wherein said attractant is selected from nitrocellulose and nylon membranes. S"

8. A method according to claim 7, wherein said membrane is positively 4 J, charged. 960815,p:\oporjms,48117.93,31 -31

9. A method according to any one of claims 4 to 8, wherein zoospores of P.cinnamomi attracted to said solid support are detected by immunoassay utilising a species-specific anti-P.cinnamomi monoclonal antibody.

A method according to claim 9 wherein said species-specific monoclonal antibody binds to antigen on the surface of P.cinnamomi cysts.

11. A solid support when used in the detection of motile soil organisms such as Phytophthera cinnamomi in a sample, said solid support being an attractant or being treated with an attractant for said organisms wherein said attractant attracts i said organisms by chemotaxis and/or electrotaxis. S.

12. A solid support according to claim 11 in the form of a dipstick.

13. A solid support according to claim 12, which is treated with an attractant selected from the group consisting of aspartic acid, glutamic acid arginine, pectin and ethanol.

14. A solid support according to claim 13, wherein said attractant is selected from nitrocellulose and nylon membrane.

A solid support according to claim 14, wherein said membrane is positively charged,

16. A kit for detecting motile soil organisms such as Phytophthera cinnamomi in a sample by a method according to any one of claims 1 to 10, which comprises a solid support according to any one of claims 11 to 15, and (ii) means for detecting organisms attracted to said solid support.

17. A kit according to claim 16, wherein said detecting means comprises means S for performing an immunoassay. 1 II 960815,p:\opor\jms,48117.93,32 32

18. A kit according to claim 17, wherein said means for performing an immunoassay comprises antibody binding to said organisms and reporter means for detecting said bound antibody.

19. A kit according to claim 16 for detecting zoospores of P.cinnamomi in a sample, wherein said detecting means comprises species-specific anti-P.cinnamomi monoclonal antibody. A kit according to claim 19, wherein said species-specific monoclonal antibody binds to antigen on the surface of P.cinnamomi cysts. *4S Dated this 14th day of February, 1997 i.The Australian National University By Its Attorneys Davies Collison Cave -c- INTERNATIONAL SEARCH REPORT A. CLASSIFICATION OF SUBJECT MATER Int. C1. 5 C12Q 1/04, 1/06, G01N 33/569 International application No. PCT/AU 93/00487 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) Int IC 5 C12Q; GO1N; CHEM ABS. KEYWORD AS BELOW Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched AU: IPC as above Electronic data base consulted during the international scar:h (name of data base, and where pracicable, search terms us;,) Derwent: MOTIL:, MOBIL:, ORGANISM#, MICROORGANISM#, CELL:, CHEMO:, ELECTRO:, ATTRACT:, ADHER: C. DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to Claim No. Experimental Mycology vol. 8, pp. 238-244, 1984, "A Method for Estimating lectin binding to fungal zoospores cysts" Mary L. Williams (et al) A whole document EP 61141 (Max-Planck-Gesellschaft zur Forderung der Wissenschaften 17 March 1982 (17.03.82) A whole document EP 214340 (Bio Control Systems Inc.) 18 March 1987 (18.03.87) A whole document F Further documents are listed See patent family annex. in the continuation of Box C. Special categories of cited documents: later document published after the international filing date or pnority date and not in conflict document definin the eneral state of the art which is with the applicationbut cited to understand the not considered to be articular relevance principle or theory underling the invention earlier document but published on or after the aocument of particular reevance; the claimed international filing date invention cannot be considered novel or cannot be document which may throw doubts on priority claim(s) considered to involve an inventive step when the or which is cited to establish the publication date of document is taken alone another citation or other special reason (as specified) document of particular relevance; the claimed document referring to an oral disclosure, use, invention cannot be considered to involve an exhibition or other means inventive step when the document is combined document published prior to the international filing date with one or more other such documents, such but later than the priority date claimed combination being obvious to a person skilled in the art document member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report December 1993 (15.12.93) flA q4 1 -<q4t) Name and mailing address of the ISA/AU Authorized officer AUSTRALIAN INDUSTRIAL PROPERTY ORGANISATION PO BOX 200 WODEN ACT 2606 AUSTRALIA yG. COLLINS Facsimile No. 06 2853929 Telephone No. (06) 2832569 Form PCT/ISA/210 (continuation of first sheet (July 1992) cophin INTERNATIONAL SEARCH P2POPT Information on patent family memb International applir'.aon No. PCT/AU 93/00487 This Annex lists the known publication level patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent Document Cited in Search Patent Family Member Report EP 61141 AU 550626 US 4514387 EP 214340 CA 1271707 US 5132229 END OF ANNEX Form PCT/ISA/210(patent family annex)(July 1992) cophin