Proteins involved in the attachment of a hyperparasite, Pasteuria penetrans, to its plant-parasitic nematode host, Meloidogyne incognita

JOURNAL

OF INVERTEBRATE

PATHOLOGY

59,

B-23

(1992)

Proteins Involved in the Attachment of a Hyperparasite, Pasteuria penetrans, to Its Plant-Parasitic Nematode Host, Meloidogyne incognita K. G. DAVIES, M. P. ROBINSON, AND V. LAIRD AFRC

Institute

of Arable

Crops

Research,

Rothumsted

Experimental

Station,

Harper&n,

Received June 25, 1990; accepted April

Proteins extracted from the surface of sporesof three populations of Pusteuria penetrans, known to exhibit different levels of attachment to Meloidoggne spp., were compared on SDS-PAGE gels. Silver staining revealed several differences between the populations. Immunoblotting with a polyclonal antibody raised against whole sporesof P. penetrans population PPl revealed a number of quantitative and qualitative differences, but most proteins were conserved. A competitive ELISA was usedto show that different populations of spores had different avidities for the antibody. In attachment assays the antibody was also found to prevent spore attachment. The results suggestthat differences in the amount and nature of the proteins on the surface of sporesmay account for differences observed in host specificity. o 1992 Academic Press,

Inc.

KEY WORDS: Meloidoggne incognita; Pasteuria pene-

tram; host specificity; biological control; Western blot; competitive ELBA.

SDS-PAGE;

INTRODUCTION

Pasteuria penetrans produces spores which adhere to the cuticle and infect many species of nematodes, of which a number are important crop pests (Sayre and Starr, 1988). The hyperparasite is a bacterium and has been observed in both tropical and temperate regions of the world but its taxonomy is unclear. Host specificity is important when classifying parasites but such studies of Pasteuria are sparse. Although attachment studies have shown that populations of the bacterium isolated from one nematode genus can adhere to another genus and even infect (Bhattacharya and Swarup, 1988), individual populations usually exhibit a restricted host range; in only one case, when 11 populations ofP. penetrans all isolated from Meloidogyne spp., were tested against a range of different cyst and rootknot nematodes, was there a small degree of spore attachment to a cyst nematode (Davies et al., 1988). Spores have been shown to differ even in their level of attachment to the same species of nematode (Stirling, 18 0022-2011/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Hertfordshire

AL5

2JQ,

United

Kingdom

10, 1991

1985). It therefore seems likely that the group consists of several species and subspecies of which two have so far been identified: P. penetrans which parasitizes Meloidogyne spp. and P. thornei which parasitizes Pratylenchus spp. (Starr and Sayre, 1988). Several new populations of Pasteuria, possibly two new groups, have been observed to parasitize and suppress populations of cyst nematodes. One group, observed to parasitize Heterodera avenae, H. cajani, H. sorghi, and H. zeae, follows a life-cycle similar to that of P. penetrans on root-knot nematodes; spores adhere to the cuticle of second-stage juveniles, germinate to produce microcolonies which proliferate throughout the pseudocoelom, and complete their life-cycle when infected females engorged with spores break open (Bhattacharya and Swarup, 1988; Sharma and Swarup, 1988). Lectins are considered important in the attachment of some nematophagous fungi to nematode cuticle (Nordbring-Hertz and Mattiasson, 1979; Jansson et al., 1985; Nordbring-Hertz, 1984; Zuckerman and Jansson, 1984). The interaction between the cuticle of secondstage juveniles of root-knot nematodes and P. penetrans is poorly understood. Early work failed to implicate lectin-carbohydrate interactions (O’Brien, 1980; Stirling et al., 1986); however, it is now thought that a lectin similar to wheat germ agglutinin (WGA) which was thought to bind to N-acetyl-D-glucosamine residues on the spore may, in part, be involved, as it has been found to inhibit attachment of spores (Bird et al., 1989). Glycoconjugates in the amphidial region of second-stage juveniles differ among Meloidogyne populations (Davis et al., 19881, and more recently it has been found that proteins isolated from juvenile body walls of different races of Meloidogyne differentially bound concanavalin A and soybean agglutinin (Davis and Kaplan, 1990). As different populations of Pasteuria spores exhibit differential levels of attachment to the cuticle of second-stage juveniles of the same and different populations of nematode (Davies et al., 1988; Stirling, 1985), it seems likely that more than one mechanism is

P. penetrans ATTACHMENT TO M. incognita

involved. The present study reports an investigation into the proteins on the surface of the spores of P. penetrans. SDS-PAGE gels of the surface proteins from three populations of P. penetruns were electro-blotted onto nitrocellulose membranes and probed with a polyclonal antibody raised against P. penetruns population PPl. The relatedness of these populations was further investigated by competitive ELISA. Finally the polyclonal antibody was used to inhibit spore attachment to M. incognita second-stage juvenile cuticle and to determine whether there were any differences in the ability of the antibody to inhibit attachment between the different Pasteuria spore populations. The results are discussed in relation to differential host specificity. MATERIALS

Nematodes

AND

METHODS

19

and a 4% (w/v> stacking

gel (pH 6.8). Spores (lo71 of P. PPl, PNG, and PCal were boiled for 5 min at 100°C in 200 ~1 of sample buffer (50 mM Tris/HCl, pH 6.8, 2% SDS w/v, 10% glycerol, 0.0025% bromphenol blue w/v, and 2% P-mercaptoethanol) and spun at 10,OOOg for 5 min, and 20 pl of the supernatant was loaded onto the stacking gel. Two replicates of each sample were run through the gel until the tracking dye reached the gel front. Prestained SDS-PAGE molecular weight markers (Sigma) were run on each gel. Following electrophoresis the gel slabs were either fixed (40% methanol/lO% acetic acid v/v) and silverstained (Bio Rad Silver Stain) or electro-blotted onto nitrocellulose filter membranes.

penetruns populations

Western Blotting

After the completion of SDS-PAGE, proteins were transferred onto nitrocellulose membranes in a continFresh infective second-stage juveniles of M. incoguous buffer (39 IIIM glycine, 48 InM Tris, 0.0375% w/v nita (Race 2 obtained from North Carolina State SDS, 10% methanol) using LKB Multiphor II NovaBlot University, Raleigh) were obtained from egg masses transfer unit. Membranes were blocked dissected from the roots of tomato (Lycopersicon escu- electrophoretic with 5% blotto (0.05% v/v Tween, 5% w/v dried Zentum) cv. Pixie. The nematode cultures were mainskimmed milk in PBS) for 60 min on a rotary shaker at tained routinely in a glasshouse at 25°C. The eggs were room temperature, washed (~2) in PBST (0.05% v/v placed on a hatching tray in tap water at room temTween in PBS) for 15 min, and then incubated with perature and allowed to hatch (Hooper, 1986). PPl polyclonal antibody at 11500 dilution in 5% blotto Pasteuria Endospores overnight on a rotary shaker at room temperature. Following incubation with the PPl antibody, they were Populations of endospores of P. penetruns (PPl obwashed (x2 as above) and then incubated with l/250 tained from S. R. Gowen, University of Reading, UK; goat anti-rabbit IgG alkaline phosphatase conjugate PNG isolated from a soil sample from Papua New (Sigma) in 5% blotto for 2 hr at room temperature. Guinea supplied by J. Bridge, CIP International InstiAfter a further two washes (as above) the membranes tute of Parasitology, St Albans, UK; PCAL obtained were incubated with alkaline phosphatase substrate from S. Verdejo, University of California, Davis), were (0.033% w/v nitro blue tetrazohum (Sigma), 0.017% routinely increased in the laboratory following the w/v 5-bromo-4-chloro-3-indolyl phosphate (Sigma)), in method of Stirling and Wachtel (1980). Spores were Tris-HCl, pH 9.5, substrate buffer (100 IIIM Tris, 100 obtained from crushed females of M. incognita that had been infected with P. penetrans. These were purified by mM NaCl, 50 mM MgClz); the reaction was terminated loading l-ml spore suspensions on a two-step sucrose with several washes in distilled water. gradient (4 ml, 40% and 2 ml 90%), spun at 25,000g at Competitive Two-Plate ELISA 4°C for 60 min); they were then washed in PBS (10 mM sodium phosphate buffer, pH 7.4, 0.9% sodium chloPlate 1: Each well of a 96-well microtiter plate ride), counted using a hemocytometer, and stored at (round-bottomed; Dynatech) was coated with 0.5 pg - 20°C. poly-L-lysine in 50 l.~l PBS for 2 hr at room temperature, washed (~2) with PBS, and blotted dry. To each Polyclonul Antibody well, 50 ~1 of a Pusteuria spore suspension of 2.5 x lo5 An antibody, raised in rabbit against spores of P. spores ml- ’ in PBS was added and incubated for 1 hr penetrans population PPl, was kindly provided by Dr at room temperature. The plate was washed in PBS A. Persidis (Department of Biochemistry, University of and blotted dry and the spores adhering to the plate Cambridge, Cambridge, CB2 l&W, UK), having been were fixed by adding 100 ~1 0.25% glutaraldehyde to purified on a protein A-Sepharose column (Persidis, each well for 3 min. The plate was washed ( x 3) in PBS 1989). and incubated overnight at 4°C with blocker (1% w/v bovine serum albumin, 0.02% sodium azide and 100 Polyacrylamide Gel Electrophoresis (SDS-PAGE) mM glycine). Plate 2: To each well of a 96-well microPolyacrylamide gel electrophoresis (Laemmli, 1970) titer plate (round-bottomed; Dynatech) 50 ~1 of PBS in the presence of sodium dodecylsulphate (SDS) was was added. In duplicate wells in column 1,50 pl of 2 x performed using a 12% (w/v) separating gel (pH 8.8) lo6 Pasteuria spores ml-’ of each of the three Pas-

20

DAVIES,

ROBINSON,

teuria populations (PPl, PNG, and PCal) was added and double diluted across the plate, columns 1 to 12. To each well in rows A to G, 50 pl of l/500 dilution of PPl polyclonal antibody was added in PBST, and 50 ~1 of l/500 dilution of preimmune serum in PBST to row H. The plate was incubated on a plate shaker for 1 hr at room temperature. The contents of plate 1 were removed rapidly, washed (X 3) in PBST, and blotted dry, followed by the addition of 50 ~1 of the contents of each well of plate 2 to the respective wells of plate 1. Plate 1 was then incubated for 30 min on a plate shaker at room temperature; the contents were removed rapidly, washed (X 3) in PBS, and blotted dry; and 50 p.1 of l/500 dilution of goat anti-rabbit alkaline phosphatase conjugate (Sigma) in 5% blotto was added to each well. The plate was then incubated for 30 min at 37”C, washed (x3) in PBST, and developed by further incubation in 0.05% w/v p-nitrophenyl phosphate disodium (Sigma) in alkaline phosphatase substrate buffer, pH 9.8 (9.7% v/v diethanolamine). The reaction was monitored by reading the absorption at 405 nm on a Titertek Multiscan MCU340 (Flow). The percentage competition was then calculated following the method of Denyer and Crowther (1986). Analysis of parallelism (ROSS, 1980) was used to determine the significance of the differences between the curves of percentage competition. Spore Inhibition

Assay

Twelve columns of a 96-well microtiter plate (flatbottomed; Dynatech) were filled with 50 ~1 of PBS. Four wells of row A were filled with 50 ~1 of Pasteuria spores at 4 x lo6 spores ml-l for each of the three populations of P. penetruns (PPl, PNG, and PCal) and double diluted down the plate from rows A to H. To each well, of 2 columns of each Pasteuria population, 25 ~1 of PPl antibody (l/250) was added in PBST, and to the remaining wells, of 2 columns of each Pasteuria population, preimmune serum (l/250 in PBST) was added. The plate was shaken for 2 hr at room temperature before the addition of 20 second-stage juveniles of M. incognita in 25 pl of PBST. The plate was shaken overnight and the next day the number of spores adhering to the juveniles was counted using a high-power microscope (X 400). Spores were counted up to a maximum of 90; thereafter it was difficult to record the number of spores accurately. The percentage reduction in spore attachment was calculated, and the analysis of parallelism (Ross, 1980) was used to determine the significance of the differences in the reduction of spore attachment.

AND LAIRD

penetrans, but differences that distinguished one population of P. penetruns from another could also be seen. Population PPl had proteins with Mr’s of approxi-

mately 72 and 5 kDa which were not present in either of the other two populations; populations PNG and PCal had proteins with MGs of approximately 190, 24, 17, and 14 kDa not present on PPl; PCal had proteins with Ml.% of approximately 170 and 36 kDa not present on either of the other two P. penetrans populations; quantitative differences could also be observed between populations (Fig. 1). There was a high degree of similarity in the populations of P. penetrans when proteins were visualized by electro-blotting onto a nitrocellulose membrane and probed with anti-PPl antibody, although even here one or two differences can be seen (Fig. 2). The Western blot, however, revealed several proteins of population PPl, most of which were common to all the populations of P. penetruns with M,.‘s of approximately 190, 86, 58, 50 and 24 kDa, which were not visualized by silver staining. The Western blot also showed quantitative differences in reactivity of the polyclonal serum, at M,.‘s of approximately 44 and 38 kDa, between the spore populations. Competitive Two-Plate ELISA

and Spore Inhibition

The attachment of spores, in a standard assay, to the cuticle of M. incognita differed between the P. penetruns populations (P < 0.001); PPl had the highest number of spores adhering to the cuticle after 24 hr followed by PNG and then PCal (Table 1). The affinities between the different populations of P. penetrans for the anti-PPl polyclonal antibody, as measured by competition between spores of population PPl and spore PPl

Kd

PNG

PCal

180116845a-

P

48-

36260

RESULTS SDS-PAGE

and Western Blotting

Silver staining showed that there were several bands of protein common to all three populations of P.

FIG. 1. SDS-PAGE gel of proteins extracted from the surface of three populations of spores ofP. penetrans (PPl, PNG, and PCal) and stained using a silver staining kit (Bio-Rad).

P. penetruns ATTACHMENT PPl

Kd

PNG

21

TO M. incognita

TABLE 2 The Slopesof the Curves and Their Displacement from the Standard Curve for Pasteuria Population PPl”

PCal

Assay

Pasteurin population

Slope

Curve displacement*

(SE)

PPl PNG PCal

- 1.1 - 0.5 -0.1

standard 0.5 -2.1

(0.4) (0.5)

PPl PNG PCal

7.1 1.5 0.9

standard - 0.69 - 1.87

(0.06) (0.05)

Percentage competition Percentage reduction 26* FIG. 2. SDS-PAGE gel of proteins extracted from the surface of three populations of spores of P. penetrans and electro-blotted onto a nitrocellulose membrane and probed with a polyclonal antibody raised against spores of P. penetrans population PPI.

populations of PNG and PCal, were significantly different (P < 0.01; Table 2, Fig. 3). The spore population PCal was the least able to compete with population PPl; the slope of the competition curve was reduced and parallel curve analysis showed that the displacement of the curve was statistically significant (P < 0.01; Table 2). The competition curve for P. penetruns spore population PNG vs PPl was only slightly different from that of the standard, PPl vs PPl (Fig. 3; Table 2). The incubation of spores in anti-PPl polyclonal antibody reduced the number of spores adhering to the cuticle compared to incubating the spores in preimmune sera (Fig. 4). The antibody had the greatest effect on reducing attachment of P. penetruns population PCal followed by PNG and had the least effect on the attachment of population PPl (Fig. 4).

a As calculated by an analysis of parallelism using MLP (Ross, 1980), of: percentage competition between Pasteuria populations PPl, PNG, and PCal for anti-PPl polyclonal antibody; and the percentage reduction in spore attachment, to the cuticle of second-stage juveniles, due to the incubation of spores in anti-PPl polyclonal antibody for each of the Pasteuria populations. * ANOVA percentage competition P < 0.01; percentage reduction P < 0.01.

ences, unless proteins of different sizes, recognized by the antibody, have common epitopes. The profiles of the Western blots suggest that the different populations of P. penetruns have many proteins in common; however, because silver staining was not sensitive enough to reveal all the proteins that may have been present, it seems likely that silver staining has underestimated the differences between the populations. Proteins under investigation here were extracted from the same number of spores (10’) for each spore population and 35 r

DISCUSSION

SDS-PAGE followed by silver staining showed that there were differences between the three populations of P. penetrans, and Western blotting revealed further proteins that were undetected by silver staining. A Western blot, probed with an antibody raised to a particular spore population, will tend to show the similarities between the populations and not show the differTABLE 1 Pasteuria SporesAttached to Second-StageJuveniles (52) of Meloidogyne incognita after Incubation in a Suspension

5-

of 5 x lo5 Sporesml- ’ PBS P. penetrans population

Mean number of spores adhering per J2*

Standard error

PPl PNG PCal

89.2 54.8 25.6

2.3 11.9 8.4

* ANOVA

P < 0.001

100

50

25 12.56.3

3.1

1.6

0.6

0.4

0.2

0.1

SPORE CONCENTRATION (x 1O4 1 FIG. 3. Percentage competition between three populations of P. _. _ penetrans tar a polyclonal antibody raised against spores of P. penetrans population PPl serially diluted from lo6 spores/ml down a microtiter plate. (-0-j PPl; (--A-) PNG; (---W---) PCal.

22

DAVIES, PPI

ROBINSON,

PNG

lOOr

REDUCTION

PCal 100

= 80 r P UI

80

2 60 0 :: iii L 40 i

60 '\

100 50

2512.5 Spore

8.3 3.1 1.6 concentration

100

50

2512.5

6.33.1

1.6

per mL (x104)

FIG. 4. The mean number of spores of three populations of P. (PPl, PNG, PCal) adhering to the cuticle of second-stage juveniles of M. incognita (n = 20) when spores, serially diluted, were incubated prior to juvenile exposure, in preimmune sera (--O-j and polyclonal antibody (-A-) raised against P. penetruns population PPl. The percentage reduction in attachment of spores due to the presence of the antibody is also given, as calculated from the respective differences in attachment between spores incubated in preimmune sera and those incubated in polyclonal antibody. (-0-j PPl; (-A-) PNG; (---m---J PCal. penetruns

the blots revealed quantitative differences between bands that were conserved, showing that different amounts of protein were present in the different populations. Antibody raised against PPl has been shown to recognize the surface of PPl spores by immunofluorescence (Davies et al., 1990). The competitive ELISA measured the relative avidity of a single antiserum to the surface of three populations of Pasteuria spores; small differences in avidity produce large changes in the competitive ability of heterologous isolates (Denyer and Crowther, 1986) and this was used to help characterize the three populations of spores. It showed that not all the P. penetrans populations had the same avidity for the antibody and indicates that there are differences in the quantities of protein present on the surface of the different spore populations recognized by

AND LAIRD

the antibody. The attachment assay showed that PCal has a lower avidity for the cuticle of M. incognita and is more easily inhibited from adhering to it by the antibody. This suggests that PCal has fewer adhesions compatible with M. incognita than either PNG or PPl; either different amounts or types of epitopes are present on the surface of the spores of different populations of P. penetrans. Some of these epitopes are probably involved in the attachment of spores to nematode cuticle. However, whether all these epitopes are directly involved is difficult to judge: it is possible that some of the proteins visualized on the Western blot leaked out from within the spores and are not involved in attachment. Our results are contrary to experiments in which spores fixed with glutaraldehyde, which cross-links proteins, still attached to nematode cuticle (Stirling et al., 1986). It is also possible that the antibody is adhering to epitopes not directly involved in attachment but is still able to inhibit adhesion. The Western blot shows that the antibody is mainly adhering to proteins common to all three P. penetruns populations. If epitopes that were not involved in attachment were being recognized there would be an equal reduction in attachment of all P. penetruns spores to nematode cuticle in the attachment assay. As there was a differential reduction in the rate of attachment between the different populations of spores, this strongly suggests that the antibody is recognizing and adhering to epitopes involved in spore attachment. In the few detailed studies of the adhesion of bacteria to plant-parasitic nematodes, P. penetrans to root-knot nematodes (Stirling, 1985; Davies et al., 1988) and corynebacterium to Anguina ugrostis (Bird, 1985), differential adhesion occurred between each of the different bacterial populations and their respective host populations. The nature by which spores of P. penetruns recognize and adhere to the cuticle of a host juveniles, and, moreover, exhibit characteristic levels of host specificity in attachment assays, is not understood. The chemical composition of the surface coat (glycocalyx) of root-knot nematodes is complex and different proteins, including collagen, are associated with different stages in nematode development (Reddigari et al., 1986; Robinson et al., 1989). Spores of P. penetrans show not only differential host specificity but also differential stage specificity; one population of spores parasitizing M. incognita was able to adhere only to second-stage juveniles (Davies, unpublished data), whereas spores of other populations have been found adhering to secondstage juveniles and males of M. acronea (Page and Bridge, 19851, Heteroderu auenae, (Sturhan, 1985) and Tylenchus semipenetrans (Fattah et al., 1989). This suggests that spores are recognizing common attachment sites present on both the second-stage juvenile cuticle and the male. This type of host parasite recognition may involve lectin-carbohydrate interactions, such as WGA which has been shown to inhibit spore

P. penetrans

ATTACHMENT

attachment (Bird et al., 1989); however, this was the only lectin found to have an effect from a number of Iectins investigated and other types of binding must be operating if the differential host and stage specificity is to be explained. The differences between the spore populations of P. penetruns in the SDS-PAGE electrophoretic profiles, the avidities for antibody, and the inhibition of attachment by the antibody to second-stage juvenile cuticle suggest that differences in proteins on the surface of the spores may in part account for differences observed in host specificity. These results, together with the possibility of lectin-carbohydrate interactions, therefore show that the nature of attachment is complex and dependent on several types of binding and recognition. The production of monoclonal antibodies to secondstage juvenile cuticle and P. penetrms spores would be a method by which the nature of adhesion could be investigated further. ACKNOWLEDGMENTS The authors thank Dr. A. Persidis, Department of Biochemistry, University of Cambridge, Tennis Court Lane, Cambridge, UK, for kindly providing the antibody. The first and final authors are grateful to the Agricultural Genetics Co. for their financial support. This work was carried out under MAFF Plant Health License No. PHF 26(X20(58). REFERENCES Bhattacharya, D., and Swarup, G. 1988. Pasteuriapenetruns a pathogen of the genus Heteroderur Its effect on nematode biology and control. Indian J. Nematol. 18, 61-70. Bird, A. F. 1985. The nature of the adhesion of Corynebacterium rathuyi to the cuticle of infective larva of Anguina agrostis. Int. J. Parusitol. 15, 301308. Bird, A. F., Bonig, I., and Bacic, A. 1989. Factors affecting the adhesion of micro-organisms to the surfaces of plant-parasitic nematodes. Parasitology 98, 155-164. Davies, K. G., Flynn, C. A., and Kerry, B. R. 1988. The life-cycle and pathology of the root-knot nematode parasite Pusteuria penetruns. In “Proceedings of the Brighton Crop Protection ConferencePests and Diseases,” Vol. III, pp. 1221-1226. Davies, K. G., Robinson, M. P., and Persidis, A. 1990. The characterisation of Pusteuria penetruns using a polyclonal antibody and its effect on spore attachment to the second-stage juvenile of Meloidogyne incognita In “Second International Nematology Congress, p. 69. Veldhoven, The Netherlands. Davis, E. L., and Kaplan, D. T. 1990. Internal and body wall glycoproteins from hatched juveniles of Meloidogyne spp. In “Second International Nematology Congress,” p. 69. Veldhoven, The Netherlands. Davis, E. L., Kaplan, D. T., Permar, T. A., Dickson, D. W., and Mitchell, D. J. 1988. Characterisation of carbohydrates on the surface of second-stage juveniles of Meloidogyne spp. J. NemutoE. 20, 609-619. Denyer, M. S., and Crowther, J. R. 1986. Use of indirect and competitive ELISAs to compare isolates of equine influenza A virus. J. Viral. Methods 14, 253-265. Fattah, F. A., Saleh, H. M., and Aboud, H. M. 1989. Parasitism of the

TO M. incognita

23

citrus nematode, Tylenchus semipenetrans by Pasteuria penetrans in Iraq. J. Nematol. 21, 431-433. Hooper, D. J. 1986. Extraction of free-living stages from soil. In “Laboratory Methods for Working with Plant and Soil Nematodes” (J. F. Southey, Ed.). MAFF, HMSO Ref. Book 402. Jannson, H.-B., Jeyaprakash, A., and Zuckerman, B. M. 1985. Differential adhesion and infection of nematodes by the endoparasitic fungus Merio coniospora (Deuteromycetes). Appl. Environ. Microbiol.

49, 552-555.

Laemmli, E. K. 19’70. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 686-685. Nordbring-Hertz, B. 1984. Mycelial development and lectin carbohydrate interactions in nematode trapping fungi. In “The Ecology and Sociology of Fungal Mycelium” (D. H. Jennings and A. D. M. Rayner, Eds.), pp. 421-432. Cambridge Univ. Press. Nordbring-Hertz, B., and Mattiason, B. 1979. Action of a nematode trapping fungus shows lectin mediated host-microorganism interaction. Nature 281, 477-479. O’Brien, P. C. 1980. Studies on parasitism of Meloidogyne jauanicu by Bacillus penetrans. J. Nematol. 12, 234. Page, S. L. J., and Bridge, J. 1985. Observations on Pasteuria penetrans as a parasite of Meloidogyne acronea. Nematologica 31,238240. Persidis, A. 1989. ‘The Biochemistry of Attachment of Pasteuria penetrans to Plant-Parasitic Nematodes.” Ph.D. thesis, University of Cambridge, Cambridge, UK. Reddigari, S. R., Jansma, P. L., Premachandran, D., and Hussey, R. S. 1986. Cuticular collagenous proteins of second-stage juveniles and adult females of Meloidogyne incognita: Isolation and partial characterization. J. Nematol. 18, 294-302. Robinson, M. P., Delgado, J., and Parkhouse, R. M. E. 1989. Characterisation of stage-specific cuticular proteins of Meloidogyne incognita by radio iodination. Physiol. Mol. Plant Pathol. 35, 135-140. Ross, G. J. S. 1980. “The Maximum Likelihood Program (M.L.P.).” AFRC Institute for Arable Crops Research, Rothamsted Experimental Station, UK. Sayre, R. M., and Starr, M. P. 1988. Bacterial diseases and antagonists of nematodes. In “Diseases of Nematodes” (G. 0. Poinar, Jr., and H. B. Jansson, Eds.), pp. 69-101. CRC Press. Sharma, R., and Swarup, G. 1988. “Pathology of Cyst Nematodes.” Malhotro, New Delhi. Starr, M. P., and Sayre, R. M. 1988. Pasteuria thornei sp. nov. and Pasteuria penetrans sensu strict0 Emend.: Mycelial and endospore forming bacteria parasitic, respectively, on plant-parasitic nematodes of the genera Pratylenchus and Meloidogyne. Ann. Inst. Pasteur/Microbiol. 139, 11-31. Stirling, G. R. 1985. Host specificity of Pasteuria penetruns within the genus Meloidogyne Nematologica 31, 203-209. Stirling, G. R., Bird, A. F., and Cakurs, A. B. 1986. Attachment of Pasteuria penetruns to the cuticles of root-knot nematodes. Reu. N@matol. 9, 251-260. Stirling, G. R., and Wachtel, M. F. 1980. Mass production ofBacillus penetruns for the biological control of root-knot nematodes. Nematologica 26, 308-312. Sturhan, D. 1985. Untersuchungen uber verbreitung und wirte des Nematodenparasiten Bacillus penetrans. In “Mitteleilungen aus der Biologischen Bundesanstalt fiir Lande und Forstwirtschaft, Berlin,” Vol. 226, pp. 75-93. Zuckerman, B. M., and Jansson, H. B. 1984. Nematode chemotaxis and possible mechanisms of host/prey recognition. Annu. Rev. Phytopathol. 22, 95-113.