Ultrastructure. could have considerable potential as a biologicle. control agent against plant nematodes. Con-

Transcription

1 JOURNAL OF BACTERIOLOGY, Feb. 1977, p Copyright 1977 American Society for Microbiology Vol. 129, No. 2 Printed in U.S.A. Bacterial Parasite of a Plant Nematode: Morphology and Ultrastructure RICHARD M. SAYRE* AND WILLIAM P. WERGIN Nematology Laboratory, Plant Protection Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland Received for publication 17 September 1976 The life cycle of a bacterial endoparasite of the plant-parasitic nematode Meloidogyne incognita was examined by scanning and transmission electron microscopy. The infective stage begins with the attachment of an endospore to the surface of the nematode. A germ tube then penetrates the cuticle, and mycelial colonies form in the pseudocoelom. Sporulation is initiated when terminal cells of the mycelium enlarge to form sporangia. A septum within each sporangium divides the forespore from the basal or parasporal portion of the cell. The forespore becomes enclosed by several laminar coats. The parasporal cell remains attached to the forespore and forms the parasporal microfibers. After the newly formed spores are released into the soil, these microfibers apparently enable a mature spore to attach to the nematode. These results indicate that the endoparasite is a procaryotic organism having structural features that are more common to members of Actinomycetales and to the bacterium Pasteuria ramosa than to the sporozoans or to the family Bacillaceae, as previous investigators have concluded. The original description of plant nematodes bacterial and can be readily cultured, they containing the parasitic disease organism Duboscqia penetrans presented a complex life cycal control agent against plant nematodes. Con- could have considerable potential as a biologicle consisting of several stages and placed the sequently, an investigation was undertaken to organism among obligate microsporidial parasites in the family Nosematidae (15). As a re- during its life cycle and (ii) determine whether (i) examine the fine structure of the organism sult, the organism was considered beyond in the organism could be grown in vitro and subjected to characterization studies for possible vitro cultivation. In addition, the proposed intricate life cycle and small size of the parasite identification. probably discouraged other investigators from further studies. Recent advances in electron MATERIALS AND METHODS microscopy have enabled investigators to more Second-stage larvae of M. incognita bearing parasitic spores were obtained from the roots of the pep- readily evaluate the taxonomic classification of organisms. As a result, Mankau and Imbriani per Capsicum annuum Linn. grown in a greenhouse soil bench. Spore-incumbered larvae were (7-9) demonstrated that this parasitic organism was procaryotic. Their observations removed separated from the soil by using a Baermann funnel or the organism from were the eucaryotic protozoans. obtained by allowing healthy larvae to migrate through the infested soil in a Baermann funnel. These infected larvae were added to the soil Furthermore, Mankau (7) compared the organism to the milky disease of insects caused by around tomato or pepper roots to maintain and increase the parasite in the greenhouse. In the labora- Bacillus popilliae Dutky Because the parasite exhibited several similarities to B. tory, the life cycle of the bacterial parasite was popilliae and formed resistant endospores, he studied during the root-knot nematode infestation of designated it B. penetrans. the roots of tomato seedlings. Seedlings were germinated and grown on blotter paper according to a Spores, similar to the type originally described by Thorne and subsequently examined method suggested by Marks and Sayre (10). Larvae by Mankau, have long been present and are bearing spores were pipetted onto the blotter paper adjacent to the roots of tomato frequently observed on the cuticles of larvae of seedlings. The blotter paper containing the inoculated seedlings was returned to trays of vermiculite. The seedlings were Meloidogyne incognita (Kofoid and White) Chitwood 1949 that are collected from greenhouse soils at Beltsville. If these spores are orescent growth lamps. Root galls caused by rootwatered with Hoagland solution and exposed to flu- 1091

2 1092 SAYRE AND WERGIN knot larvae were harvested from the tomato seedlings on days 2 to 25, prepared for electron microscopic studies, and examined to follow the development of the bacterial parasite within the root-knot females. Tissue for electron microscopic observation was prepared according to the method of Endo and Wergin (5). Briefly, root galls were placed on a sheet of dental wax containing several drops of 3% glutaraldehyde in 0.05 M phosphate buffer (ph 6.8). Galls were cut into 2- to 3-mm segments and transferred to glass vials containing glutaraldehyde and buffer. In addition, mature parasitized females about 30 days old were handpicked from roots and crushed in molten 3% agar at 50 C. This procedure allowed the spores to disperse for easier observation. The agar was allowed to cool and solidify and then was placed in 3% glutaraldehyde. Rinsing and postfixation in osmium tetroxide also were carried out in 0.05 M phosphate buffer. Fixation in glutaraldehyde for 1.5 h was followed by washing in six changes of buffer over a period of 1 h. The galls and agar, containing crushed females, were postfixed in 2% osmium tetroxide for 2 h, dehydrated in an acetone series, and infiltrated with a low-viscosity medium (13). Silvergray sections of selected galls and nematodes were cut on a Sorvall MT-2 ultramicrotome with a diamond knife and mounted on uncoated copper grids (75 by 100 mesh). The sections were stained with 2% aqueous uranyl acetate (10 min) and then with lead citrate (5 min). Thin sections were viewed with a Philips 200 electron microscope operating at 60 kv with 20-,um apertures. Larvae containing the bacterial spores were prepared for scanning electron microscopic examination either by chemically fixing with 3% glutaraldehyde in 0.05 M phosphate buffer for 1.5 h, dehydrating in an ethanol series, and critically point drying or by crushing the. parasitized females onto the surface of an aluminum stub and air drying. Aluminum stubs containing the dried specimens were coated with gold-palladium. Larvae and females were examined with a Hitachi HHS-2R scanning electron microscope operating at 15 or 20 kv. J. BACTERIOL. RESULTS The parasites found adhering to the cuticle of root-knot larvae bear structural similarities to bacterial spores. Therefore, in this study, the parasite is referred to as a "bacterial spore parasite of nematodes" (BSPN), and bacteriological terminology is used to describe the structural features of the spore. Mature spores. Spores measuring about 3.8,um and adhering to the surface of root-knot larvae are considered mature. Two distinct forms of these spores can be observed with the scanning electron microscope. The surface of one form appears as a wrinkled membrane that encompasses the entire spore (Fig. 1). This "tmembrane" is the exosporium, which is generally sloughed prior to germination. In the absence of the exosporium, the spore can be resolved into two distinct components: a central endospore, 2.3,um in diameter, that is spherical, and a peripheral matrix, 0.5 gm wide, that surrounds the endospore (Fig. 2). The smooth central surface of the endospore is easily distinguishable from its peripheral matrix, which forms an encircling ring with a particulate surface. Cross sections viewed with the transmission electron microscope reveal that the endospore consists of a central, highly electron-opaque core that is surrounded by an inner and an outer wall composed of several distinct layers (Fig. 3). When observed with the transmission electron microscope, the peripheral matrix of the spore is fibrillar. Fine microfibrillar strands, about 1.5 nm thick, arc outward and downward from the sides of the endospore to the cuticle of the nematode, where they become more electron dense. External and perpendicular to the microfibrillar matrix are short "hairs." The particulate surface of the matrix observed with the scanning electron microscope is formed by these hairs projecting outward (Fig. 2). Germinating spores. A mature spore attaches to the surface of a nematode so that a basal ring of wall material lies flatly against the cuticle. A median section through the endospore and perpendicular to the surface of the nematode bisects this basal ring. As a result, the ring appears as two protruding pegs, which are continuous with the outer layer of the spore wall, and rests on the cuticular surface of the nematode (Fig. 3). The peripheral fibers of the spore are also closely associated with the cuticle. The fibers, which encircle the endospore, lie along the surface of the nematode and follow the irregularities of the cuticular annuli. They do not appear to penetrate the cuticle (Fig. 3). Germination of the endospores apparently occurs after the spore-encumbered nematode enters the root and initiates feeding in the host. Even when these conditions occur, only 20% of the spores encountered within roots showed any structural changes indicative of germination. As a result, germinating spores are difficult to locate, and the detailed changes that occur during germination are difficult to document. The germ tube of the endospore emerges through the central opening of the basal ring (Fig. 4). The emerging tube penetrates the cuticle of the nematode and enters the hypodermal tissue. The germ tube, which measures 0.24,um in diameter, appears to have walls similar in electron opacity and structure to those of the

3 VOL. 129, 1977 BACTERIAL PARASITE OF A PLANT NEMATODE 1093 FIG. 1 and 2. Scanning electron micrographs of endospores associated with second-stage larvae of M. incognita. The spores are apparently attached to the cuticle along the lateral fields of the larvae. At this stage, the spores occasionally retain the exosporium, which results in the appearance of a crinkled surface (Fig. 1). When this membrane is sloughed, the central endospore (E) can be distinguished from the peripheral parasporal fibers (F in Fig. 2). x14,000. inner layers of the endospore. No structural deformations of the nematode cuticle are obvious, indicating that the germ tube exerts no appreciable force on the cuticle during this process; however, the cuticle frequently exhibits an intense electron density at this stage. Vegetative growth. Hyphae were initially encountered beneath the cuticle of the nematode near the site of germ tube penetration (Fig. 5). From this site, they apparently penetrate the hypodermal and muscle tissues and enter the pseudocoelom. Mycelial colonies up to 20,um in diameter are formed in the pseudocoelom, where they are observed after the diseased larvae penetrate plant roots (Fig. 7). The hyphae comprising the colony are septate. A hyphal cell, which is 0.20 to 0.24,um by 4.0 to 10.0,um, is bounded by a compound wall, 0.12 um thick, composed of an outer and an inner membrane (Fig. 7). The outer membrane frequently contains short projections that result in a clear space or halo, which is apparent when hyphae lie in an electron-opaque matrix (Fig. 6). The inner membrane of the wall forms the septations and delineates individual cells. In addition, this membrane is continuous with a membrane complex or mesosome that is frequently associated with the septum (Fig. 7). Sporulation. Sporulation is a synchronously initiated process that involves the terminal hyphal cells of the mycelium. As the process begins, the size of the terminal cell enlarges. Its shape becomes ovate, and the structure and content of the cytoplasm change from a granular matrix, which contains numerous ribosomes as found in the hyphal cells, to one that lacks particulate organelles (Fig. 8). During these changes, the developing sporangia separate from their parental hyphae, which cease to grow and eventually degenerate. After these early structural alterations, a membrane forms within the sporangium and separates the upper third of the cell or forespore from its lower or parasporal portion (Fig. 9). The granular area confined within the membrane then condenses into an electron-opaque

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5 VOL. 129, 1977 body, 0.6,um in diameter (Fig. 10), which eventually becomes encircled by a multilayered wall (Fig. 11). This discrete structure is the endospore. Coincident with the formation of an endospore is the emergence of parasporal fibers. These fine fibers, which form around the base of the spore, differentiate from an electrontranslucent granular substance (Fig. 10). They appear to connect with and radiate from the external layer of the wall of the endospore (Fig. 11 to 14). During development of the parasporal fibers, the formation of another membrane, the exosporium, isolates the newly formed endospore within the sporangium (Fig. 13 and 14). At this later stage of spore development, the granular content of the paraspore becomes less dense (Fig. 13), and degenerates and disappears (Fig. 14). As a result, the mature sporangium contains a fully developed endospore enclosed within the exosporium (Fig. 14). The cell wall of the sporangium remains intact until the nematode is disrupted and the endospores are released. The exosporium apparently remains associated with the endospore until contact is made with a new nematode and the infection cycle restarts. BACTERIAL PARASITE OF A PLANT NEMATODE DISCUSSION Life cycle. The life cycle of BSPN consists of three distinct stages: spore germination, vegetative growth, and sporulation (Fig. 15). The germination of spores is the most elusive stage to document because of the technical difficulties associated with locating and sectioning germinating spores that are attached to the surface of a nematode embedded in root tissue. However, the evidence obtained in our study indicates that a germinating spore produces the germ tube that penentrates the cuticle of the larva. The germ tube evidently gives rise to vegetative hyphae. This concept is supported by the occurrence of small filamentous hyphal colonies in the hypodermal tissue, directly beneath the cuticle of the nematode. Eventually, these vegetative hyphae reach the pseudocoelom and totally colonize the nematode. For extensive colonization to occur, primary colonies, which arise from a few infection sites, must be capable of generating daughter colonies. The occurrence of degenerating hyphal cells and cell walls in the older mycelium suggests that fragmentation may be partially involved in the colonization process. Fragmentation would allow hyphae to break away from the parent colony and become fully established at a more distant site within the nematode. Fragmentation, as well as degeneration, also appears to occur during the early stages of sporulation. During this stage, the terminal hyphal cells begin to increase in size, becoming separated from the hyphae, and differentiate into spore-producing structures. Further development of the spores results in about a 50-fold increase in the size of the original mycelial cell. This process occurs within the mature female. As a result of the extensive vegetative growth of hyphae and the cell enlargement associated with spore maturation, a single female nematode is lysed and releases mature spores, which are estimated to exceed 2 million (7). Similarities between BSPN and other parasitic spore diseases. (i) Spore diseases of nematodes. The life cycle and morphology of BSPN are similar to those described for other parasitic spore diseases of nematodes. For example, Thorne observed a spore disease of nematodes as early as 1940 (15). Sufficient evidence was not obtained to properly classify this parasite. However, the organism was believed to be a protozoan and was named Dubosciqia penetrans. In 1960, Williams reexamined and established the life cycle of D. penetrans (16). Although he did not entirely agree with the protozoan classification, the name remained unchanged. More recently, Mankau has examined a spore disease of nematodes believed to be identical to that caused by D. penetrans. However, his electron microscopic examination indicated that the organism may be a procaryote; therefore, Mankau has suggested reclassifica- FIG. 3 to 14. Series of transmission electron micrographs illustrating the developmental stages in the life cycle of BSPN. FIG. 3. Cross section through an endospore on the surface of a nematode. Parasporal fibers (F) appear to radiate outward from the lower half of the spore to the cuticle of the nematode. On the surface offibers, short "hairs" (arrows) project outward to give the reticulated surface appearance shown in Fig. 2. No obvious structural attachments can be traced into the cuticle of the nematode. However, the spore is apparently sufficiently secure to displace the cortical cells (C) of the plant as the nematode penetrates the root. x36,000. FIG. 4. Cross section through a germinated spore. The penetrating germ tube follows a sinuous path as it traverses the cuticle (C) and hypodermis (H) of the nematode. Consequently, the penetrating structure does not lie within the plane of the section for its entire length. No obvious mechanical distortion of the cuticle is 1095 associated with a germ tube; however, an electron-opaque area directly beneath a germinating spore was frequently observed. x28,000.

6 FIG. 5. Section through vegetative hyphae that lie beneath the cuticle (C) of a nematode. After cuticular penetration by the germ tube, vegetative hyphae are formed and traverse the hypodermal tissue as they grow toward the pseudocoelom. x22,000. FIG. 6. Section through hyphae surrounded by a granular matrix in the pseudocoelom of a nematode. The hyphae contain numerous ribosomes and amorphous areas (arrow), which may contain genetic material. Hyphae are bounded by a compound wall consisting of a double membrane. Short projections, which become evident on the outer surface of hyphae that lie within an electron-opaque matrix, result in the appearance of a clear surrounding "halo" (H). x54,000. FIG. 7. Portion of a mycelial colony in the pseudocoelom of the nematode. The hyphae, which are septate, appear to bifurcate (arrows) at the margins of the colony. x24,

7 IC.\~~~~~~~~~~~m ' *1r4 *;S7S *zw 11 ;:SI s -.~~~V... Downloaded from go I #' f 11.i.4 on September 28, 2018 by guest FIG. 8. Portion of a colony that has begun to sporulate. The terminal cells have enlarged into ovate structures that eventually will separate from the parental hyphae and become sporangia. The older hyphal cells cease to grow and degenerate. x30,

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9 VOL. 129, 1977 tion of this organism as Bacillus penetrans (7). The organism we have examined in our study is similar in size and morphology to that of D. penetrans described by Thorne. Its stages in the life cycle resemble those illustrated by Williams (16), and the organism has fine structural features similar to those of B. penetrans described by Mankau (7). Therefore, we conclude that all of these organisms, D. penetrans, B. penetrans, and BSPN, are probably the same species. However, for reasons that will be subsequently discussed, neither D. penetrans nor B. penetrans seems to be the appropriate designation for the organism at this time. (ii) Spore disease of Daphnia. In addition to its similarities to the spore disease of nematodes, the life cycle of BSPN also closely resembles the developmental stages of the bacterium Pasteuria ramosa. This organism, which was originally described by Metchnikoff (11) in 1888, is a parasite of Daphnia magna and D. pulex. Metchnikoff recognized and drew the endospores and their formation for Pasteuria. His drawings of these stages are strikingly similar to those of BSPN illustrated in Fig. 15. These morphological similarities suggest that BSPN may be related to the bacterial genus Pasteuria Ċlassification. Although precise classification based on structural features of the organism and elucidation of its life cycle cannot be made, our observations allow us to partially clarify the current confusion. For example, observations of the fine structure indicate that the organism is a procaryote. Therefore, it would not be properly classified among the protozoans in the genus Duboscqia, as previously suggested. This conclusion agrees with that of Mankau, who placed the pathogen in the genus Bacillus (7). However, a comparison of the features exhibited by B. popilliae, an accepted Bacillus, and BSPN indicates that several significant features distinguish these organisms. A mature spore of BSPN lacks a sporangial wall, a crystalline parasporal body, and an exosporium. These structures are present in the BACTERIAL PARASITE OF A PLANT NEMATODE 1099 mature spore of B. popilliae. Germinating spores of BSPN form germ tubes, extensive mycelial growth, and sporangia, whereas B. popilliae gives rise to a vegetative rod, with continued growth occurring by binary fission. Finally, the mycelial growth pattern exhibited by BSPN is a feature not generally attributed to Bacillaceae. Although the genus Pasteuria could have been an attractive possibility for the classification of BSPN, this genus was recently reevaluated (6), and the new description offered by Staley (14) may no longer be applicable to the organism. Alternatively, the BSPN bears several structural features that are more characteristic of the Actinomycetales. The structural features of BSPN compare favorably to those of a thermoactinomycete, Thermoactinomyces vulgaris, which was initially described by Tsiklinsk (1899). Both organisms have dense spore coats that confer resistance to heat and desiccation. In addition, both organisms germinate by the formation of a mycelial germ tube. Because BSPN exhibits mycelial growth and forms endospores, the organism may be an actinomycete. Recently, Cross (1) stated that many authors describing new endospore-forming species tended to place organisms in either Bacillus or Clostridium. As an alternative, he and his coworkers (1-4) have established a few genera within Actinomycetales where they believe some true endospore-forming species should be placed. We believe that BSPN may belong among these genera. This suggestion is based on the morphology and ultrastructural features of the organism. For example, the procaryotic nature of this organism coupled with its branching filamentous mycelia, which are less than 1,um in diameter, are characteristics of Actinomycetales. Additionally, the organism has a double-track wall bearing hairlike projections similar to that of several species of the anaerobic actinomycetes described by Slack (12). Although our observations lead us to suggest FIG. 9. Section through a sporangium that has separated from the parental hypha. Part ofa membrane has formed and separates the anterior third ofthe spore or forespore from the lower parasporal segment. x48,000. FIG. 10. Median section through a sporangium illustrating an early stage of endospore development. An electron-opaque body has formed within the forespore. Loosely surrounding the body is a membrane that will condense and contribute to the multilayered wall of the mature endospore. Near the base of the forming spore are two lateral electron-translucent areas (arrows), which will develop into the parasporal fibers. x38,000. FIG. 11. Sporangium containing a partially formed endospore. At this stage, the developing wall does not completely surround the endospore. The lateral regions, which will differentiate into the parasporal fibers, have enlarged and caused the sporangium to increase in width. x26,000. FIG. 12. Section through a sporangium containing an endospore that is nearly fully developed. The spore, whose multilayered wall has formed, begins to pull away from the wall of the sporangium (arrow) and further distends its lateral walls. x28,000.

10 'S 21. * rrw;m.c.. \ X. E. a-,*~ i.~i '!,'U s.*n 7 s ' e~~~~~~~a, s ''di'rk >.s~~ t~~1 FIG. 13. Section through a sporangium undergoing the final stages of differentiation. The endospore loses its tight apposition with the wall of the sporangium, and the matrix of the parasporal segment becomes coagulated, coarsely granular, and electron opaque. x32,000. FIG. 14. Median section through a sporangium containing a fully developed endospore. The last stages of differentiation of the endospore include the formation of an encircling membrane or exosporium (E) and the emergence of parasporal fibers (arrows) within the granular material that lies laterally around the spore. x38,

11 VOL. 129, 1977 *: -..o / is\{~\1.l 1*ODI\ BACTERIAL PARASITE OF A PLANT NEMATODE 1101,... ' , lia} IUAIl. ~ ~ ~ h F A.. ~~~~~( )1 FIG. 15. Life cycle ofthe bacterial spore parasite of nematodes. that BSPN is a member of Actinomycetales, confirmation and precise classification will depend on cultivating the organism in vitro and collecting spores in sufficient quantities to analyze their cell wall composition and to determine their nucleotide base ratio. ACKNOWLEDGMENTS We acknowledge the technical assistance of Pamela Lloyd, Barbara Wei, W. A. Habicht, and R. B. Ewing, who prepared Fig. 15. Appreciation is also extended to R. Faust, D. Farr, and T. G. Pridham, for suggesting future experimental procedures and reviewing this manuscript, and, in particular, to R. E. Davis for calling our attention to the genus Pasteuria. LITERATURE CITED 1. Cross, T The diversity of bacterial spores. J. Appl. Bacteriol. 33: Cross, T., F. L. Davies, and P. D. Walker Thermoactinomyces vulgaris. I. Fine structure of the developing endospores, p In A. N. Barker, G. W. Gould, and J. Wolf (ed.), Spore research. Academic Press Inc., New York. 3. Cross, T., and J. Lacey Studies on the genus Thermomonospora, p In H. Prauser (ed.), The Actinomycetales. The Jena International Symposium on Taxonomy. Gustav Fisher Verlag, Jena. 4. Cross, T., P. D. Walker, and G. W. Gould Thermophilic actinomycetes producing resistant endospores. Nature (London) 220: Endo, B. Y., and W. P. Wergin Ultrastructural investigation of clover roots during early stages of it.. i.fi.. kk ",....% F infection by the root-knot nematode, Meloidogyne incognita. Protoplasma 78: Hirsch, P Re-evaluation of Pasteuria ramosa Metchnikoff 1888, a bacterium pathogenic for Daphnia species. Int. J. Syst. Bacteriol. 22: Mankau, R Bacillus penetrans n. comb. causing a virulent disease of plant-parasitic nematodes. J. Invertebr. Pathol. 26: Mankau, R Prokaryote affinities of Duboscqia penetrans, Thorne. J. Protozool. 21: Mankau, R., and J. L. Imbriani The life cycle of an endoparasite in some Tylenchid nematodes. Nematologica 21: Marks, C. F., and R. M. Sayre The effect of potassium on the rate of development of the root-knot nematodes Meloidogyne incognita, M. javanica, and M. hapla. Nematologica 10: Metchnikoff, M. E Pasteuria ramosa, un representant des bacteries a division longitudinale. Ann. Inst. Pasteur Paris 2: Slack, J. M., and M. A. Gerencser Actinomyces, filamentous bacteria; biology and pathogenicity, p Burgess Publishing Co.., Minneapolis. 13. Spurr, A A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26: Staley, J. T Budding bacteria of the Pasteuria- Blastobacter group. Can. J. Microbiol. 19: Thorne, G Dubosecqia penetrans n. sp. (Sporozoa, Microsporidia, Nosematidae), a parasite of the nematode Pratylenchus pratensis (de Man) Filipjev. Proc. Helminthol. Soc. Wash. 7: Williams, J. R Studies on the nematode soil fauna of sugarcane fields in Mauritius 5. Notes upon a parasite of root-knot nematodes. Nematologica 5: