Bacterial Parasite of Plant Nematode: Morphologyand · Bacterial Parasite ofa Plant Nematode:...

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JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 1091-1101 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 20705 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 investi- gators have concluded. The original description of plant nematodes containing the parasitic disease organism Du- boscqia penetrans presented a complex life cy- cle consisting of several stages and placed the organism among obligate microsporidial para- sites in the family Nosematidae (15). As a re- sult, the organism was considered beyond in vitro cultivation. In addition, the proposed in- tricate life cycle and small size of the parasite probably discouraged other investigators from further studies. Recent advances in electron microscopy have enabled investigators to more readily evaluate the taxonomic classification of organisms. As a result, Mankau and Imbriani (7-9) demonstrated that this parasitic organism was procaryotic. Their observations removed the organism from the eucaryotic protozoans. Furthermore, Mankau (7) compared the organ- ism to the milky disease of insects caused by Bacillus popilliae Dutky 1940. Because the parasite exhibited several similarities to B. popilliae and formed resistant endospores, he designated it B. penetrans. Spores, similar to the type originally de- scribed by Thorne and subsequently examined by Mankau, have long been present and are frequently observed on the cuticles of larvae of Meloidogyne incognita (Kofoid and White) Chitwood 1949 that are collected from green- house soils at Beltsville. If these spores are bacterial and can be readily cultured, they could have considerable potential as a biologi- cal control agent against plant nematodes. Con- sequently, an investigation was undertaken to (i) examine the fine structure of the organism during its life cycle and (ii) determine whether the organism could be grown in vitro and sub- jected to characterization studies for possible identification. MATERIALS AND METHODS Second-stage larvae of M. incognita bearing para- sitic spores were obtained from the roots of the pep- per Capsicum annuum Linn. grown in a green- house soil bench. Spore-incumbered larvae were separated from the soil by using a Baermann funnel or were obtained by allowing healthy larvae to mi- grate through the infested soil in a Baermann fun- nel. These infected larvae were added to the soil around tomato or pepper roots to maintain and in- crease the parasite in the greenhouse. In the labora- tory, the life cycle of the bacterial parasite was studied during the root-knot nematode infestation of the roots of tomato seedlings. Seedlings were germi- nated and grown on blotter paper according to a method suggested by Marks and Sayre (10). Larvae bearing spores were pipetted onto the blotter paper adjacent to the roots of tomato seedlings. The blotter paper containing the inoculated seedlings was re- turned to trays of vermiculite. The seedlings were watered with Hoagland solution and exposed to flu- orescent growth lamps. Root galls caused by root- 1091 on June 24, 2020 by guest http://jb.asm.org/ Downloaded from

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JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 1091-1101Copyright © 1977 American Society for Microbiology

Vol. 129, No. 2Printed in U.S.A.

Bacterial Parasite of a Plant Nematode: Morphology andUltrastructure

RICHARD M. SAYRE* AND WILLIAM P. WERGINNematology Laboratory, Plant Protection Institute, Agricultural Research Service, U.S. Department of

Agriculture, Beltsville, Maryland 20705

Received for publication 17 September 1976

The life cycle of a bacterial endoparasite of the plant-parasitic nematodeMeloidogyne incognita was examined by scanning and transmission electronmicroscopy. The infective stage begins with the attachment of an endospore tothe surface of the nematode. A germ tube then penetrates the cuticle, andmycelial colonies form in the pseudocoelom. Sporulation is initiated whenterminal cells of the mycelium enlarge to form sporangia. A septum within eachsporangium divides the forespore from the basal or parasporal portion of the cell.The forespore becomes enclosed by several laminar coats. The parasporal cellremains attached to the forespore and forms the parasporal microfibers. Afterthe newly formed spores are released into the soil, these microfibers apparentlyenable a mature spore to attach to the nematode. These results indicate thatthe endoparasite is a procaryotic organism having structural features that aremore common to members of Actinomycetales and to the bacterium Pasteuriaramosa than to the sporozoans or to the family Bacillaceae, as previous investi-gators have concluded.

The original description of plant nematodescontaining the parasitic disease organism Du-boscqia penetrans presented a complex life cy-cle consisting of several stages and placed theorganism among obligate microsporidial para-sites in the family Nosematidae (15). As a re-sult, the organism was considered beyond invitro cultivation. In addition, the proposed in-tricate life cycle and small size of the parasiteprobably discouraged other investigators fromfurther studies. Recent advances in electronmicroscopy have enabled investigators to morereadily evaluate the taxonomic classification oforganisms. As a result, Mankau and Imbriani(7-9) demonstrated that this parasitic organismwas procaryotic. Their observations removedthe organism from the eucaryotic protozoans.Furthermore, Mankau (7) compared the organ-ism to the milky disease of insects caused byBacillus popilliae Dutky 1940. Because theparasite exhibited several similarities to B.popilliae and formed resistant endospores, hedesignated it B. penetrans.

Spores, similar to the type originally de-scribed by Thorne and subsequently examinedby Mankau, have long been present and arefrequently observed on the cuticles of larvae ofMeloidogyne incognita (Kofoid and White)Chitwood 1949 that are collected from green-house soils at Beltsville. If these spores are

bacterial and can be readily cultured, theycould have considerable potential as a biologi-cal control agent against plant nematodes. Con-sequently, an investigation was undertaken to(i) examine the fine structure of the organismduring its life cycle and (ii) determine whetherthe organism could be grown in vitro and sub-jected to characterization studies for possibleidentification.

MATERIALS AND METHODSSecond-stage larvae ofM. incognita bearing para-

sitic spores were obtained from the roots of the pep-per Capsicum annuum Linn. grown in a green-house soil bench. Spore-incumbered larvae wereseparated from the soil by using a Baermann funnelor were obtained by allowing healthy larvae to mi-grate through the infested soil in a Baermann fun-nel. These infected larvae were added to the soilaround tomato or pepper roots to maintain and in-crease the parasite in the greenhouse. In the labora-tory, the life cycle of the bacterial parasite wasstudied during the root-knot nematode infestation ofthe roots of tomato seedlings. Seedlings were germi-nated and grown on blotter paper according to amethod suggested by Marks and Sayre (10). Larvaebearing spores were pipetted onto the blotter paperadjacent to the roots of tomato seedlings. The blotterpaper containing the inoculated seedlings was re-turned to trays of vermiculite. The seedlings werewatered with Hoagland solution and exposed to flu-orescent growth lamps. Root galls caused by root-

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1092 SAYRE AND WERGIN

knot larvae were harvested from the tomato seed-lings on days 2 to 25, prepared for electron micro-scopic studies, and examined to follow the develop-ment of the bacterial parasite within the root-knotfemales. Tissue for electron microscopic observationwas prepared according to the method of Endo andWergin (5). Briefly, root galls were placed on a sheetof dental wax containing several drops of 3% glutar-aldehyde in 0.05 M phosphate buffer (pH 6.8). Gallswere cut into 2- to 3-mm segments and transferred toglass vials containing glutaraldehyde and buffer. Inaddition, mature parasitized females about 30 daysold were handpicked from roots and crushed in mol-ten 3% agar at 50°C. This procedure allowed thespores to disperse for easier observation. The agarwas allowed to cool and solidify and then was placedin 3% glutaraldehyde. Rinsing and postfixation inosmium tetroxide also were carried out in 0.05 Mphosphate buffer. Fixation in glutaraldehyde for 1.5h was followed by washing in six changes of bufferover a period of 1 h. The galls and agar, containingcrushed females, were postfixed in 2% osmium te-troxide for 2 h, dehydrated in an acetone series, andinfiltrated with a low-viscosity medium (13). Silver-gray sections of selected galls and nematodes werecut on a Sorvall MT-2 ultramicrotome with a dia-mond 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 leadcitrate (5 min). Thin sections were viewed with aPhilips 200 electron microscope operating at 60 kVwith 20-,um apertures.

Larvae containing the bacterial spores were pre-pared for scanning electron microscopic examina-tion either by chemically fixing with 3% glutaralde-hyde in 0.05 M phosphate buffer for 1.5 h, dehydrat-ing in an ethanol series, and critically point dryingor by crushing the. parasitized females onto the sur-face of an aluminum stub and air drying. Aluminumstubs containing the dried specimens were coatedwith gold-palladium. Larvae and females were ex-amined with a Hitachi HHS-2R scanning electronmicroscope operating at 15 or 20 kV.

RESULTSThe parasites found adhering to the cuticle of

root-knot larvae bear structural similarities tobacterial spores. Therefore, in this study, theparasite is referred to as a "bacterial spore par-asite of nematodes" (BSPN), and bacteriologi-cal terminology is used to describe the struc-tural features of the spore.Mature spores. Spores measuring about 3.8

,um and adhering to the surface of root-knotlarvae are considered mature. Two distinctforms of these spores can be observed with thescanning electron microscope. The surface ofone form appears as a wrinkled membrane thatencompasses the entire spore (Fig. 1). This"tmembrane" is the exosporium, which is gener-ally sloughed prior to germination. In the ab-sence of the exosporium, the spore can be re-

solved into two distinct components: a centralendospore, 2.3 ,um in diameter, that is spheri-cal, and a peripheral matrix, 0.5 gm wide, thatsurrounds the endospore (Fig. 2). The smoothcentral surface of the endospore is easily distin-guishable from its peripheral matrix, whichforms an encircling ring with a particulate sur-face.

Cross sections viewed with the transmissionelectron microscope reveal that the endosporeconsists of a central, highly electron-opaquecore that is surrounded by an inner and anouter wall composed of several distinct layers(Fig. 3).When observed with the transmission elec-

tron microscope, the peripheral matrix of thespore is fibrillar. Fine microfibrillar strands,about 1.5 nm thick, arc outward and downwardfrom the sides of the endospore to the cuticle ofthe nematode, where they become more elec-tron dense. External and perpendicular to themicrofibrillar matrix are short "hairs." Theparticulate surface of the matrix observed withthe scanning electron microscope is formed bythese hairs projecting outward (Fig. 2).Germinating spores. A mature spore at-

taches to the surface of a nematode so that abasal ring of wall material lies flatly againstthe cuticle. A median section through the endo-spore and perpendicular to the surface of thenematode bisects this basal ring. As a result,the ring appears as two protruding pegs, whichare continuous with the outer layer of the sporewall, and rests on the cuticular surface of thenematode (Fig. 3).The peripheral fibers of the spore are also

closely associated with the cuticle. The fibers,which encircle the endospore, lie along the sur-face of the nematode and follow the irregulari-ties of the cuticular annuli. They do not ap-pear to penetrate the cuticle (Fig. 3).Germination of the endospores apparently oc-

curs after the spore-encumbered nematode en-ters the root and initiates feeding in the host.Even when these conditions occur, only 20% ofthe spores encountered within roots showedany structural changes indicative of germina-tion. As a result, germinating spores are diffi-cult to locate, and the detailed changes thatoccur during germination are difficult to docu-ment.The germ tube of the endospore emerges

through the central opening of the basal ring(Fig. 4). The emerging tube penetrates the cuti-cle of the nematode and enters the hypodermaltissue. The germ tube, which measures 0.24 ,umin diameter, appears to have walls similar inelectron opacity and structure to those of the

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BACTERIAL PARASITE OF A PLANT NEMATODE

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 peripheralparasporal fibers (F in Fig. 2). x14,000.

inner layers of the endospore. No structuraldeformations of the nematode cuticle are ob-vious, indicating that the germ tube exerts noappreciable force on the cuticle during thisprocess; however, the cuticle frequently ex-hibits an intense electron density at this stage.

Vegetative growth. Hyphae were initiallyencountered beneath the cuticle of the nema-tode near the site of germ tube penetration(Fig. 5). From this site, they apparently pene-trate the hypodermal and muscle tissues andenter the pseudocoelom. Mycelial colonies up to20 ,um in diameter are formed in the pseudocoe-lom, where they are observed after the diseasedlarvae penetrate plant roots (Fig. 7).The hyphae comprising the colony are sep-

tate. A hyphal cell, which is 0.20 to 0.24 ,um by4.0 to 10.0 ,um, is bounded by a compound wall,0.12 um thick, composed of an outer and aninner membrane (Fig. 7). The outer membranefrequently contains short projections that re-sult in a clear space or halo, which is apparentwhen hyphae lie in an electron-opaque matrix

(Fig. 6). The inner membrane of the wall formsthe septations and delineates individual cells.In addition, this membrane is continuous witha membrane complex or mesosome that is fre-quently associated with the septum (Fig. 7).

Sporulation. Sporulation is a synchronouslyinitiated process that involves the terminal hy-phal cells of the mycelium. As the process be-gins, the size of the terminal cell enlarges. Itsshape becomes ovate, and the structure andcontent of the cytoplasm change from a granu-lar matrix, which contains numerous ribo-somes as found in the hyphal cells, to one thatlacks particulate organelles (Fig. 8). Duringthese changes, the developing sporangia sepa-rate from their parental hyphae, which cease togrow and eventually degenerate.

After these early structural alterations, amembrane forms within the sporangium andseparates the upper third of the cell or foresporefrom its lower or parasporal portion (Fig. 9).The granular area confined within the mem-brane then condenses into an electron-opaque

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BACTERIAL PARASITE OF A PLANT NEMATODE

body, 0.6 ,um in diameter (Fig. 10), which even-tually becomes encircled by a multilayered wall(Fig. 11). This discrete structure is the endo-spore.

Coincident with the formation of an endo-spore is the emergence of parasporal fibers.These fine fibers, which form around the baseof the spore, differentiate from an electron-translucent granular substance (Fig. 10). Theyappear to connect with and radiate from theexternal layer of the wall of the endospore (Fig.11 to 14). During development of the parasporalfibers, the formation of another membrane, theexosporium, isolates the newly formed endo-spore within the sporangium (Fig. 13 and 14).At this later stage of spore development, thegranular content of the paraspore becomes lessdense (Fig. 13), and degenerates and disappears(Fig. 14). As a result, the mature sporangiumcontains a fully developed endospore enclosedwithin the exosporium (Fig. 14).The cell wall of the sporangium remains in-

tact until the nematode is disrupted and theendospores are released. The exosporium ap-parently remains associated with the endosporeuntil contact is made with a new nematode andthe infection cycle restarts.

DISCUSSIONLife cycle. The life cycle of BSPN consists of

three distinct stages: spore germination, vege-tative growth, and sporulation (Fig. 15). Thegermination of spores is the most elusive stageto document because ofthe technical difficultiesassociated with locating and sectioning germi-nating spores that are attached to the surface ofa nematode embedded in root tissue. However,the evidence obtained in our study indicatesthat a germinating spore produces the germtube that penentrates the cuticle of the larva.The germ tube evidently gives rise to vegeta-tive hyphae. This concept is supported by theoccurrence of small filamentous hyphal coloniesin the hypodermal tissue, directly beneath thecuticle of the nematode. Eventually, these veg-

etative hyphae reach the pseudocoelom and to-tally colonize the nematode. For extensive colo-nization to occur, primary colonies, which arisefrom a few infection sites, must be capable ofgenerating daughter colonies. The occurrenceof degenerating hyphal cells and cell walls inthe older mycelium suggests that fragmenta-tion may be partially involved in the coloniza-tion process. Fragmentation would allow hy-phae to break away from the parent colony andbecome fully established at a more distant sitewithin the nematode.

Fragmentation, as well as degeneration, alsoappears to occur during the early stages of spor-ulation. During this stage, the terminal hyphalcells begin to increase in size, becoming sepa-rated from the hyphae, and differentiate intospore-producing structures. Further develop-ment of the spores results in about a 50-foldincrease in the size of the original mycelial cell.This process occurs within the mature female.As a result of the extensive vegetative growthof hyphae and the cell enlargement associatedwith spore maturation, a single female nema-tode is lysed and releases mature spores, whichare estimated to exceed 2 million (7).

Similarities between BSPN and other para-sitic spore diseases. (i) Spore diseases of ne-matodes. The life cycle and morphology ofBSPN are similar to those described for otherparasitic spore diseases of nematodes. For ex-ample, Thorne observed a spore disease of ne-matodes as early as 1940 (15). Sufficient evi-dence was not obtained to properly classify thisparasite. However, the organism was believedto be a protozoan and was named Dubosciqiapenetrans. In 1960, Williams reexamined andestablished the life cycle of D. penetrans (16).Although he did not entirely agree with theprotozoan classification, the name remainedunchanged. More recently, Mankau has exam-ined a spore disease of nematodes believed to beidentical to that caused by D. penetrans. How-ever, his electron microscopic examination in-dicated 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 lifecycle ofBSPN.

FIG. 3. Cross section through an endospore on the surface of a nematode. Parasporal fibers (F) appear toradiate 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 obviousstructural attachments can be traced into the cuticle of the nematode. However, the spore is apparentlysufficiently 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 ittraverses the cuticle (C) and hypodermis (H) of the nematode. Consequently, the penetrating structure doesnot lie within the plane of the section for its entire length. No obvious mechanical distortion of the cuticle isassociated with a germ tube; however, an electron-opaque area directly beneath a germinating spore was

frequently observed. x28,000.

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FIG. 5. Section through vegetative hyphae that lie beneath the cuticle (C) of a nematode. After cuticularpenetration by the germ tube, vegetative hyphae are formed and traverse the hypodermal tissue as they growtoward the pseudocoelom. x22,000.

FIG. 6. Section through hyphae surrounded by a granular matrix in the pseudocoelom ofa nematode. Thehyphae contain numerous ribosomes and amorphous areas (arrow), which may contain genetic material.Hyphae are bounded by a compound wall consisting ofa double membrane. Short projections, which becomeevident on the outer surface of hyphae that lie within an electron-opaque matrix, result in the appearance ofaclear 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. x24,000.

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FIG. 8. Portion of a colony that has begun to sporulate. The terminal cells have enlarged into ovatestructures that eventually will separate from the parental hyphae and become sporangia. The older hyphalcells cease to grow and degenerate. x30,000.

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BACTERIAL PARASITE OF A PLANT NEMATODE

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 thelife cycle resemble those illustrated by Wil-liams (16), and the organism has fine structuralfeatures similar to those of B. penetrans de-scribed by Mankau (7). Therefore, we concludethat all of these organisms, D. penetrans, B.penetrans, and BSPN, are probably the samespecies. However, for reasons that will be sub-sequently discussed, neither D. penetrans norB. penetrans seems to be the appropriate desig-nation for the organism at this time.

(ii) Spore disease of Daphnia. In addition toits similarities to the spore disease of nema-todes, the life cycle of BSPN also closely resem-bles the developmental stages of the bacteriumPasteuria ramosa. This organism, which wasoriginally described by Metchnikoff (11) in1888, is a parasite of Daphnia magna and D.pulex. Metchnikoff recognized and drew the en-dospores and their formation for Pasteuria. Hisdrawings of these stages are strikingly similarto those of BSPN illustrated in Fig. 15. Thesemorphological similarities suggest that BSPNmay be related to the bacterial genus Pasteu-ria.

Classification. Although precise classifica-tion based on structural features of the orga-nism and elucidation of its life cycle cannot bemade, our observations allow us to partiallyclarify the current confusion. For example, ob-servations of the fine structure indicate thatthe organism is a procaryote. Therefore, itwould not be properly classified among the pro-tozoans in the genus Duboscqia, as previouslysuggested. This conclusion agrees with that ofMankau, who placed the pathogen in the genusBacillus (7). However, a comparison of the fea-tures exhibited by B. popilliae, an acceptedBacillus, and BSPN indicates that several sig-nificant features distinguish these organisms.A mature spore of BSPN lacks a sporangialwall, a crystalline parasporal body, and an exo-sporium. These structures are present in the

mature spore of B. popilliae. Germinatingspores of BSPN form germ tubes, extensivemycelial growth, and sporangia, whereas B.popilliae gives rise to a vegetative rod, withcontinued growth occurring by binary fission.Finally, the mycelial growth pattern exhibitedby BSPN is a feature not generally attributedto Bacillaceae.Although the genus Pasteuria could have

been an attractive possibility for the classi-fication of BSPN, this genus was recently re-evaluated (6), and the new description offeredby Staley (14) may no longer be applicable tothe organism. Alternatively, the BSPN bearsseveral structural features that are more char-acteristic of the Actinomycetales. The struc-tural features of BSPN compare favorably tothose of a thermoactinomycete, Thermoactino-myces vulgaris, which was initially describedby Tsiklinsk (1899). Both organisms have densespore coats that confer resistance to heat anddesiccation. In addition, both organisms germi-nate by the formation of a mycelial germ tube.Because BSPN exhibits mycelial growth andforms endospores, the organism may be an ac-tinomycete.Recently, Cross (1) stated that many authors

describing new endospore-forming speciestended to place organisms in either Bacillus orClostridium. As an alternative, he and his co-workers (1-4) have established a few generawithin Actinomycetales where they believesome true endospore-forming species should beplaced. We believe that BSPN may belongamong these genera. This suggestion is basedon the morphology and ultrastructural featuresof the organism. For example, the procaryoticnature of this organism coupled with itsbranching filamentous mycelia, which are lessthan 1 ,um in diameter, are characteristics ofActinomycetales. Additionally, the organismhas a double-track wall bearing hairlike projec-tions similar to that of several species of theanaerobic 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 hasformed 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. Anelectron-opaque body has formed within the forespore. Loosely surrounding the body is a membrane that willcondense and contribute to the multilayered wall of the mature endospore. Near the base ofthe forming sporeare 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 notcompletely 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 ofthe sporangium (arrow) and furtherdistends its lateral walls. x28,000.

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FIG. 13. Section through a sporangium undergoing the final stages of differentiation. The endospore losesits tight apposition with the wall of the sporangium, and the matrix of the parasporal segment becomescoagulated, coarsely granular, and electron opaque. x32,000.

FIG. 14. Median section through a sporangium containing a fully developed endospore. The last stages ofdifferentiation of the endospore include the formation of an encircling membrane or exosporium (E) and theemergence of parasporal fibers (arrows) within the granular material that lies laterally around the spore.x38,000.

1100

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Page 11: Bacterial Parasite of Plant Nematode: Morphologyand · Bacterial Parasite ofa Plant Nematode: Morphologyand Ultrastructure RICHARD M. SAYRE* AND WILLIAM P. WERGIN NematologyLaboratory,

BACTERIAL PARASITE OF A PLANT NEMATODE 1101*: - -., liA} IUAIl.~ ~ ~ h F A

.. ~~~~~()1..o / is\{~\1 .l 1*ODI\

FIG. 15. Life cycle ofthe bacterial spore parasite of nematodes.

that BSPN is a member of Actinomycetales,confirmation and precise classification will de-pend on cultivating the organism in vitro andcollecting spores in sufficient quantities to ana-lyze their cell wall composition and to deter-mine their nucleotide base ratio.

ACKNOWLEDGMENTSWe acknowledge the technical assistance of Pamela

Lloyd, Barbara Wei, W. A. Habicht, and R. B. Ewing, whoprepared Fig. 15. Appreciation is also extended to R. Faust,D. Farr, and T. G. Pridham, for suggesting future experi-mental procedures and reviewing this manuscript, and, inparticular, to R. E. Davis for calling our attention to thegenus Pasteuria.

LITERATURE CITED1. Cross, T. 1970. The diversity of bacterial spores. J.

Appl. Bacteriol. 33:95-102.2. Cross, T., F. L. Davies, and P. D. Walker. 1971. Ther-

moactinomyces vulgaris. I. Fine structure of the de-veloping endospores, p. 175-179. In A. N. Barker,G. W. Gould, and J. Wolf (ed.), Spore research. Aca-demic Press Inc., New York.

3. Cross, T., and J. Lacey. 1970. Studies on the genusThermomonospora, p. 211-219. In H. Prauser (ed.),The Actinomycetales. The Jena International Sym-posium on Taxonomy. Gustav Fisher Verlag, Jena.

4. Cross, T., P. D. Walker, and G. W. Gould. 1968. Ther-mophilic actinomycetes producing resistant endo-spores. Nature (London) 220:352-354.

5. Endo, B. Y., and W. P. Wergin. 1973. Ultrastructuralinvestigation of clover roots during early stages of

infection by the root-knot nematode, Meloidogyne in-cognita. Protoplasma 78:365-379.

6. Hirsch, P. 1972. Re-evaluation of Pasteuria ramosaMetchnikoff 1888, a bacterium pathogenic forDaphnia species. Int. J. Syst. Bacteriol. 22:112-116.

7. Mankau, R. 1975. Bacillus penetrans n. comb. causing avirulent disease of plant-parasitic nematodes. J. In-vertebr. Pathol. 26:333-339.

8. Mankau, R. 1975. Prokaryote affinities of Duboscqiapenetrans, Thorne. J. Protozool. 21:31-34.

9. Mankau, R., and J. L. Imbriani. 1975. The life cycle ofan endoparasite in some Tylenchid nematodes. Ne-matologica 21:89-94.

10. Marks, C. F., and R. M. Sayre. 1964. The effect ofpotassium on the rate ofdevelopment of the root-knotnematodes Meloidogyne incognita, M. javanica, andM. hapla. Nematologica 10:323-327.

11. Metchnikoff, M. E. 1888. Pasteuria ramosa, un repre-sentant des bacteries a division longitudinale. Ann.Inst. Pasteur Paris 2:165-170.

12. Slack, J. M., and M. A. Gerencser. 1975. Actinomyces,filamentous bacteria; biology and pathogenicity, p.175. Burgess Publishing Co.., Minneapolis.

13. Spurr, A. 1969. A low-viscosity epoxy resin embeddingmedium for electron microscopy. J. Ultrastruct. Res.26:31-43.

14. Staley, J. T. 1973. Budding bacteria of the Pasteuria-Blastobacter group. Can. J. Microbiol. 19:609-614.

15. Thorne, G. 1940. Dubosecqia penetrans n. sp. (Sporozoa,Microsporidia, Nosematidae), a parasite of the nema-tode Pratylenchus pratensis (de Man) Filipjev. Proc.Helminthol. Soc. Wash. 7:51-53.

16. Williams, J. R. 1960. Studies on the nematode soilfauna of sugarcane fields in Mauritius 5. Notes upona parasite of root-knot nematodes. Nematologica5:37-42.

VOL. 129, 1977

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