Biocontrol: penetrans and Related Parasites of Nematodes 1 R. M. Sayre 2 Abstract: Bacillus penetrans Mankau, 1975, previously described as Duboscqia penetrans Thorne 1940, is a candidate agent for biocontrol of nematodes. This review considers the life stages of this bacterium: vegetative growth phase, colony fragmentation, sporogenesis, soil phase, spore attachment, and penetration into larvae of root-knot nematodes. The morphology of the microthallus colonies and the unusual external features of the spore are discussed. Taxonomic affinities with the actinomycetes, particularly with the genus , are considered. Also dis- cussed are other soil bacterial species that are potential biocontrol agents. Products of their bacterial fermentation in soil are toxic to nematodes, making them effective biocontrol agents. Key Words: Duboscqia, Pasteuria ramosa, Pseudomonas denitrificans, Clostridium butyricum, DesulJovibrio desulJuricans, Bacillus thuringiensis, rickettsia.

Nematodes and are two impor- tional data on two other bacterial parasites tant members of the total biota in soil of nematodes are presented briefly. The habitats. The diversity of the species, and second interaction, amensalism, considers their ubiquity and abundance in soils, for the inltibitory or antibiotic effect of some thousands o[ years have provided oppor- species of bacteria on species of plant- tunity for the evolution of intimate and parasitic nematodes. complex interactions between the two When Thorne (30) described Duboscqia groups of organisms. Theoretically, re- penetrans as a protozoan, he could not peated analysis of soil habitats for bacteria- have realized its bacterial nature because nematode interactions should reveal all of electron-microscope techniques were not the several possible interactions that could available to him, and the concept of the occur between the two species. Several have prokaryotic cell had not been introduced. been suggested by Odum and Odum (22): Later, Williams (32) studied the same or- neutralism, competition, mutualism, proto ganism in a population of root-knot females cooperation, commensalism, predation, par- taken from sugarcane, presented an interpre- asitism, and amensalism. Only two of these tation of its life stages, and indicated some interactions, parasitism and amensalism, are reservations about Thorne's identification. considered here. These interactions are most Nevertheless, he used Thorne's designation pertinent to the biological control of plant- Duboscqia. His drawings agree well with parasitic nematodes, the general topic of recent electron micrographs of the organ- this paper. isms (Fig. 1). Canning (3) also doubted The first interaction, parasitism, will the identification as a protozoan and emphasize the bacterium Bacillus penetrans stressed the organism's fungal character- Mankau, 1975 (16), and its interactions istics. Electron-microscope studies of Man- with a few plant-nematode hosts. Aspects of kau (16) and Imbriani and Mankau (11) this interaction may seem atypical because established the prokaryotic and bacterial of the bacterium's unusual morphology. nature of the organism. However, it is currently the best docu- mented example of an interaction between Bacillus penetrans--life cycle a plant nematode and parasitic bacterium A) Spore germination: Germination oc- (5,7,11,14,15,16,17,19,20,25,30,32,33). Addi- curs about 8 days after the spore- encumbered nematode enters the root and Received for publication 10 October 1979. begins feeding in the host. The germ tube ISymposium paper presented at the annual meeting of the Society of Nematologists, Salt Lake City, Utah, U.S.A., of the spore emerges through the central 23-26 July 1979. opening of the basal ring (Fig. 2) and pene- 2Research Plant Pathologist, Nematology Laboratory, Plant Protection Institute, Agricultural Research, Science trates the cuticle of the nematode. After the and Education Administration, U.S. Department of Agri- hypodermal tissue is entered, a spherical culture, Beltsville Agricultural Research Center (West), Beltsville, MD 20705. vegetative hyphal colony is formed (Fig. 3). The author gratefully acknowledges the help of Dr. While spore germination and colony forma- W. P. Wergin. Mr. R. B. Ewing, and Ms. A. Ostericher in preparing the figures. tion seem more typical of fungi than of 260 Parasites of Nematodes: Sayre 261

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Fig. I. Drawings of Bacillus penetrans from Meloidogyne incognita (left column) are compared with those of Williams (32) for Dubosqia penetrans from M. incognita and M. javanica (center column), and with those of Metchnikoff (21) for Pasteuria ramosa, pulex and D. magna (right column). Life stages of B. penetrans, based on electron micrographs, start at the top of the column with the vegetative colony, followed by daughter colonies, quartets of sporangia, doublets, single sporangium, and finally the mature endospore within the old sporangial wall at the bottom. Drawings of D. penetrans, selected from the original publica- tion, are arranged arbitrarily to show the similarities in morphology to B. penetrans, its synonomous species. Drawings of P. ramosa are placed in order of their occurrence in the life cycle of the parasite as reported by Metchnikoff (21). bacteria, close examination of the vegetative stages occurring in the nematode host, cell reveals bacterial characteristics. MeIoidogyne incognita, Sayre and Wergin B) Vegetative stage: Mankau (15) found (25) also found organelles characteristic the vegetative cells to be prokaryotic, recog- only o[ the prokaryotic cell. In neither of nized the organism's bacterial character- these studies were nuclear membranes, istics, and named the organism B. pene- plastids, mitochondria, or any other exclu- trans. In a later study involving all life sively eukaryotic cell characteristic found. 262 Journal of Nematology, Volume 12, No. 4, October 1980

Fig. 2. Cross-section through a germinated spore. The penetrating germ tube follows a sinuous path as it traverses the cuticle and hypodermis of the nematode. )<20,000. Fig. 3. Portion of a mycelial colony in the pseudocoelom of the nematode. The hyphae, which are septate, appear to bifurcate at margins of the colony. X7,500. Fig. 4. Hyphal cells are bounded by a compound wall consisting of a double membrane (arrow). X23,000. Fig. 5. Section through vegetative hyphae. The hyphae contain numerous ribosomes and amorphous areas (arrows) that may contain genetic material. 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). X 11,000. Parasites of Nematodes: Sayre 263 Clearly, the research findings indicate that D) Sporogenesis: The external morphol- tile use of tile generic protozoan designation ogy of the endospore of B. penetrans is of Duboscqia is not warranted. unique; but its internal stages of spore The bacterial hyphal cells comprising formation are typical of other endogenous the colony are septate and bounded by a sporeforming bacteria. The spore stages compound wall (Fig. 4). The outer wall consist of 1) septum formation in the an- membrane frequently contains short pro- terior of the spore mother cell; 2) condensa- jections, resulting in a clear space or halo tion of a forespore from the anterior about the mycelium (Fig. 5). The inner protoplast; 3) formation of multilayered membrane forms tile septations and de- walls about the forespore; 4) lysis of the old lineates individual cells. In addition, sporangial wall; and 5) release of an endo- mesosomes are often found associated with spore that resists heat and desiccation, and the inner membranes. A lighter, amorphous survives for long periods in storage (Fig. 7). area in the cells may contain the genetic When the vegetative and sporangial stages materials (Fig. 6). of B. penetrans are examined, they pose a C) Fragmentation of colonies: Daughter problem in systematics. The vegetative colonies are formed when intercalary cells stages, being hyphalike, suggest actinomy- lyse, allowing a separation to occur within cetous affinities, while the sporangial stages, the mother colony.The process of internal identical to spore development in the genus lysis occurs periodically during the parasite's Bacillus and Clostridium sp., suggest af- vegetative development. Gradually, daugh- finities with members of the Bacillaceae. ter colonies contain fewer, but larger, The problem would be partially resolved if vegetative cells. Eventually, quartets of de- endogenous spore formation were to occur veloping sporangia predominate in the in the Actinomycetales. Good evidence sug- nematode's psuedocoloem. These structures gests that endospores are found in some are followed by doublets of sporangia, and groups of the actinomycetes. Cross (4) pre- finally the single sporangial stages that give sented evidence for true endogenous spore rise internally to single endospores (Fig. 1). formation in several genera of the Actino-

Fig. 6. Section through microcolony. A few intercalary cells lyse or separate from one another (arrow), and this allows for the formation of daughter colonies. These processes can be found in all stages of devel- opment; ultimately only separate sporangial cell are found in the mature parasitized root-knot female. X 8,250. 264 Journal o/ Nematology, Volume 12, No. 4, October 1980

GENERALIZED BACTERIAL SPORE FORMATION

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FOIIMATION FOI~MATION VII OIETATIVIE FOR|SPORE OF OF ENDOSPORE GeOWTH SPORE CORTEX COATS

SPORE FORMATION IN BACILLUS PENETRAN5

Fig. 7. Drawings showing generalized bacterial spore formation (upper row) are compared with the sporogenous stages of Bacillus penetrans in the lower row. Aside from differences in parasporal structures, stages in the development of B. penetrans result in spores having characteristics very similar to those of the other endogenous sporeforming bacteria.

mycetales. B. penetrans also has some addi- those of some species of actinomycetes de- tional characteristics consistent with the scribed by Slack and Gerencser (28). 4) Actinomycetales: 1) Vegetative cells measure Germination of spores of B. penetrans is less than 0.2 /~m in diameter and are gram- like that o£ some endospores found in the positive. 2) Hyphal-like colonies are formed Actinomycetales (Fig. 8). and segmented during vegetative growth to While the above characteristics are indi- yield individual club-shaped sporangia. 3) cative that B. penetrans is related to mem- Ultrastructure of the cell walls reveals a bers of the Actinomycetales, the only double-tracked membrane. The outer mem- positive way to establish such a relationship brane bears hairlike projections similar to nmst be based on standard bacteriological

BACTERIAL SPORE PAgASITB B. POPILLA£ 7*, VULGARI$ OF NEMATODES /.---Procoplast -.-~.. }

~-$,porangiu m-'-~ A}am hra|poral bod~

Z Ill ff

Fig. 8. Comparison of coat morphology and germination of mature spores of B. penetrans (A), B. popillae (C), and Thermoactinomyces vulgaris (E). B. penetrans (B) and T. vulgaris (F) differ in size, but both form a filamentous tube on germination. B. popillae (D) germinates as a vegetative rod. Parasites of Nematodes: Sayre 265 tests. These tests depend on in vitro cultiva- posed for nematode attachment to occur tion of the organism, which will have to be (Fig. 9). When mature parasitized females a future research goal. are manually crushed in laboratory exami- The rediscovery of Pasteuria ramosa nation, most of their spores are surrounded Metchnikoff, 1888, by Sayre et al. (26) and by a sporangial wall and a thin exosporium. its morphological similarities to B. pene- These two covering layers are apparently trans add more difficulty to systematic removed or degraded in soil by an unknown placement of B. penetrans. B. penetrans process. and P. ramosa form microcolonies that give Because no methods are available for rise by fragmentation to quartets, then cultivation or isolation of B. penetrans from sporangia, and finally an endogenous spore soils on a selective medium, a bioassay within the old mother cell wall (Fig. 1). method is used to detect spores. Healthy The life stages in the two organisms, when larvae of the nematodes are allowed to examined by scanning and transmission migrate through spore-infested soil. The electron microscopy, are remarkably similar larvae emerging from a soil sample are ex- in sequence, suggesting a generic relation- amined microscopically for the spore load ship. However, another neotype already carried on their cuticles. The percentage of bears the name P. ramosa (ATCC No. larvae encumbered with spores and relative 27377). It has been placed by Staley (29) in numbers of spores on each larva serve as an the budding-bacteria group. The amended indicator of the concentration of spores in description of the P. ramosa neotype encom- soil. The raised lateral fields of the larvae passes a nonparasitic flagellated bacterium. are frequently observed to be the cuticular Consequently, the current neotype species areas where spore attachement occurs most of P. ramosa is unlike the original bac- frequently (Fig. 10). Another area for terium figured and described by Metchni- future research is an explanation of the koff (21). This systematic problem involving specificity of spore attachment to certain the two type strains of P. ramosa must be nematodes and not others. resolved if the generic name Pasteuria is Dutky (5) used the bioassay method of ever to be applied to tile nematode parasite. spore attachment to study the host ranges E) Soil phase: In a laboratory analysis, of three different isolates of the spore para- Mankau (16) found about 2.1 × 10~ spores site. One isolate occurred on M. incognita in each parasitized root-knot female. But and the other two adhered to populations the fate of these spores when released from of Pratylenchus brachyurus (Godfrey) from a decomposed dead nematode is not known. Florida and Maryland. The root-knot nema- Neither the distance they move nor their tode isolate of the bacterium did not attach rate of movement in soil has been measured. to the Pratylenchus sp. bacterial isolate, or If the spores are nonmotile and have no sur- vice versa. Dutky also found that spores face charge, their dispersion from point of attaching to the root-knot nematodes were release would depend largely on the rate of significantly larger in diameter than those water percolation in soil, on the size of soil from P. brachyurus. Apparently, different pore openings, on tillage practices, and to strains of B. penetrans exist. Dutky and a lesser extent, on the activities of soil in- Sayre (7) also studied the influence of soil vertebrate populations. If these spores be- moisture, texture (pore space), and tem- have as Dutky (6) described for spores of perature on spore attachment. No correla- Bacillus popillae, and are tightly bound to tion was found between the numbers of soil particles at the point of release, the spores attached per larva and the soil mois- relative importance of soil factors in move- ture level, or the soil pore size. To deter- ment of the spores would be altered. mine the thermal inactivation point of F) Spore attachment to nematode: As spores in soil, M. incognita larvae were ex- far as known, the dispersed spores in soil posed to a spore-infested soil incubated attach only to nematode hosts, atlhough previously for 1 hr at 40, 60, 80, 100, 120, edaphic factors may influence the attach- and 140 C. The level of spore attachment ment process. For example, the adhesion was no lower in soils heated to 40 C than in fibers of the mature endospore must be ex- unheated control soils. Heating soils to 60, 266 Journal of Nematology, Volume 12, No. 4, October 1980

Fig. 9. Section through a sporangium undergoing the final stage of ditIerentation. The endospore has lost its tight apposition with the wall of the sporangium. The matrix of the parasporal segment becomes finely filamentous, and parasporal fibers are found. The spore and fibers are encircled by a membrane or exosporium (E) and the old sporangial wall (w).)< 1,300.

- -- 80, 100, and 120 C reduced the number of spores attached per larva and the percentage of larvae with spores attached. No spores attached at 140 C. Spores heated to 60, 80, 100, and 120 C may be able to attach to nematodes but not to penetrate or develop inside them. The true thermal inactivation point for these spores is somewhat lower than 140 C. Investigations of the influence of other factors, such as soil pH and osmotic potential on the ability of spores to infect nematodes, would also be useful. Bacillus penetrans-- geographical distribution Parasitism of nematodes by B. penetrans has been reported from 12 states and 16 foreign countries (5). These reports prob- ably reflect the presence of a nematologist in that state or country where nematode populations are periodically sampled. They suggest worldwide distribution. Thorne (30) found 66 % of a South Carolina population Fig. 10. The three spores attached to the cuticle of Pratylenchus infested, while only 28% of this larva renew the life cycle of Bacillus pene- Of a Georgia population had succumbed to trans. The raised lateral field (F) of the larva pro- vides a favorable cuticular area for attachment, the parasite. He believed the parasite to be ×1,000. widespread in the South Atlantic states. Parasites of Nematodes: Sayre 267 Williams (32) reported that 34% of a popu- there were only a few spores per larva. In lation of root-knot nematodes were para- C, 7% were lightly infected. This result sitized by an organism very much like B. suggested that small amounts of spore- penetrans. In Florida, Esser and Sobers (9) infested soils were an effective means for observed 25% of a fixed population of introducing the parasites into field plots. Helicotylenchus to be diseased, while 80% Mankau and Prasad (19) tested seven of living populations were infested extern- nematicides at recommended field doses to ally or internally by the parasite. Other determine their compatibility with B. pene- reports were less precise. In California, trans: 1,3-D, aldicarb, carbofuran, phenami- Allen (2) reported that a high percentage of phos, and ethroprop had no noticeable effect Dolichodorus obtusus Allen were infested. on the bacterial parasite, while 1,2-dibromo- 3-chloropropane was only slightly toxic. Bacillus penetrans-- The ability of B. penetrans to prevent control experiments reproduction and eventually kill root-knot nematodes as well as several other pest nem- Mankau (16) presented data on the con- atode species, offers a good possibility for trol of root-knot nematodes using B. pene- biological control of a major crop pest. The trans. In greenhouse tests, air-dried soil resistance of these long-lived spores to heat infested with spores of B. penetrans was and desiccation, and their compatibility planted with tomato seedlings to which with nematicides, are characteristics well 10,000 root-knot nematode larvae had been suited for use in field soils. added. After 70 days, plants in the air-dried spore-infested soil had greater dry weights, Other Prokaryotic Parasites more leaves, and less root galling than plants in soil free of spores or growing in Two additional prokaryotic species are sterilized soil (Table 1). also parasitic to plant nematodes and func- In microplot experiments, Mankau (14) tion as biocontrol agents. Adams and used the following treatments: A) air-dried Eichenmuller (1) found the bacterium soil containing spores was placed in holes Pseudomonas denitrillcans Bergy et al. para- 3 inches wide and 6 inches deep; B) seed- sitizing populations of Xiphinerna ameri- lings were grown in spore-infested soil and canum Cobb, 1913. In juveniles, the bac- then transplanted into microplots; C) terium was found throughout the body, 240,000 larvae encumbered with spores were being sparse in the esophageal region. In added to plots to a depth of 4 inches. When adult females, bacteria were concentrated the soils were bioassayed 11 months after in the intestines and ovaries. This distribu- cropping, 98 % of the larvae emerging from tion suggested the possibility of transovarial treatment A were heavily encumbered with transmission. The bacterium was wide- spores. In B, only 53% carried spores, but spread, and the researchers believed that it might be a cause of difficulty in rearing Table 1. Effect of Bacillus penetrans on root- Xiphinerna species in the greenhouse. knot disease of tomato 70 days after inoculation with 10,000 MeIoidogyne incognita larvae." A second prokaryote is found in cyst nematode species: Heterodera goettingiana Liebscher from England, and Globodera Gall rostochiensis Wollenweber from Bolivia, by Leaves/ rating Tops Shepherd et al. (27); and in H. glycines Ichinohe from the United States, by Endo Soil plant (0-25) (g) A. Air-dried, 21.0(b)b 6.5(b) 5.32(b) (8). This intracellular rickettsia appears to spore-infested ( ---2.2) ( 4- 2.9) ( ± 0.8) be identical in the three cyst populations B. Sterilized, 14.20) 20.5(a) 3.120) from widely separated places. spore-infested (±4.4) (+5.2) (±0.5) Because this rickettsial organism is C. Air-dried, 13.20) 22.7(a) 3.42(a) uninfested (control) (± 4.4) (4- 4.5) ( +_0.95) morphologically similar to a companion symbiote of a leaf hopper, there is some doubt that it is a parasite. It is unicellular •Taken in part from data of Mankau 06). ~'Means followed by the same letter are not sig- and rod-shaped, and divides by binary nificantly different from each other at the 5% level. fission (Fig. 11). The cell mean length is 268 Journal o[ Nematology, Volume 12, No. 4, October 1980 stepwise degradation of soil organic matter. The products released by the metabolic activity of the bacteria vary from complex to the simplest of molecules. Some of these many products accumulate in soils and may be toxic, antibiotic, or inhibitory to plant- parasitic nematodes. Johnston (12) found that the reduction in populations of Tylenchorhynchus mar- tini Fielding, 1956, was caused by volatile fatty acids in water-saturated soil. He iso- lated a bacterium, Clostridium butyricum Prazmowski, 1880, that produced a mixture of formic, acetic, propionic, and butyric acids in its culture filtrate, which was toxic to the nematode. Hydrogen sulfide occurs in flooded rice fields in amounts apparently sufficient to control some nematodes of this crop (24). Rodriguez-Kabana et al. (24) identified Desul[ovibrio desul[uricans (Beijerinck, 1895) Kluyver and van Nell, 1936, in the "reduced" soil zone as the bacterium re- sponsible for the release of H2S. During the natural decomposition of plant residues, ammonifying bacteria ap- parently produce enough NH~ to influence nematodes. Mankau and Minteer (18) sug- gested that the NH3 produced during the decomposition of a fish amendment was probably responsible for the decline of root-knot nematodes, and Walker (31) also Fig. II. Section through a rickettsia shows a por- believed that the decomposition of nitro- tion oF the fascicle (F) of rods or tubules attached genous substances during ammonification by their ends to the plasma membrane. X126,000. and nitrification was probably responsible for the decrease in nematode populations. 1.8 ~m, and its width is 0.4 ~m. The rickettsial cell contains peculiar inclusions More recently (10), a thermostable toxin in the form of hollow rods that appear to be of Bacillus thuringiensis Berliner was found attached to the cytoplasmic membrane. Par- to be toxic to populations of Meloidogyne, allel clusters of the rods are found lying in Panagrellus, and Aphelenchus and pre- bundles, termed fascicles. In general, the vented M. incognita larvae from forming organism has only minor pathological effects galls on tomato roots. This toxin, being on cells. Many of the microorganisms within active also against many species of inverte- a cell appear to be surrounded by several brates, may lack the specificity needed for membranes of endoplasmic reticulum. This selective control of pests. Several years ago membrane enclosure may function to iso- Katznelson et al. (13) found a species of late the microorganism from the host Myxobacteriura that had the ability to lyse cytoplast. species of Caenorhabditis, Rhabditis, and Panagrellus. When tests were conducted on plant-parasitic forms, however, the isolate Amensalism-bacterial antibiosis was not lyric. Perhaps a bacterium will be Many soil bacteria species are capable found that will selectively lyse plant- of decomposing plant and animal residue. parasitic forms. If not, genetic engineering A succession of these bacteria facilitates might be used to alter bacterial cells, cans- Parasites of Nematodes: Sayre 269 ing them to be more lytic to plant-parasitic Insect Pathology an Advanced Treatise Volume 2, nematodes, or the yield of nematicidal pp. 75-114, ed. E. A. Steinhaus, Academic Press, metabolite might be increased so that they New York, 689 pp. 7. Dutky, E. M., and R. M. Sayre. 1978. Some become better biocontrol agents. factors affecting infection on nematodes by the bacterial spore parasite Bacillus penetrans. J. CONCLUSIONS Nematol. 10:285 (Abstr.). 8. Endo, B. Y. 1979. The ultrastructure and Many problems must be solved before distribution of an intracellular bacterium-like micro- microorganisms can be used to control organism in tissues of larvae of the soybean cyst nematode, Heterodera glycines. J. Ultrastruct. Res. plant-parasitic nematodes. Criteria for se- 67:1-14. lection include virulence of bacterium to 9. Esser, R. P., and E. K. Sobers. 1964. Natural nematode, susceptibility of nematodes to enemies of nematodes. Proc. Soil & Crop Sci. Soc. of the parasite, compatibility of the bacterium Florida 24:326-353. 10. Ignoffo, C. M., and V. H. Dropkin. 1977. when used with other chemical treatments, Deleterious effects of the thermostable toxin of storage or shelf life of the bacterium, and Bacillus thuringiensis on species of soil-inhabiting, time of application of bacterium for max- myceliophagous, and plant-parasitic nematodes. J. imum pest control. Obviously, judicious Kans. Entomol. Soc. 50:394-398. 11. hnbriani, J. L., and R. Mankau. 1977. Ultra- selection and planning is needed. The structure of the nematode pathogen, Bacillus pene- chances of success will probably be en- trans. J. Invertebr. Pathol. 30:337-347. hanced by computer technology. A com- 12. Johnston, T. M. 1959. Effect of fatty acids puter program prepared by Perry (23) mixtures on the rice styler nematode (Tylenchorhyn- simulates a population model for the effect chus martini Fielding, 1956). Nature 183:1392. 13. Katznelson, H., D. C. Gillespie, and F. D. of parasitic fungi on numbers of the cereal Cook. 1964. Studies on the relationship between cyst nematode, H. avenae. Work on this nematodes and other soil microorganisms. Can. ]'. model suggests the importance and direct Micrebiol. 10:699-704. effect of rainfall on the fungus infection rate 14. Mankau, R. 1972. Utilization of parasites and predators in nematode pest management ecology. in nematodes in well-drained soils. Further Proc. Tall Timbers Conf. on Ecological Animal work should improve parameter estimates, Control by Habitat Management 4:129-143. aid in evaluation of the model, and identify 15. Mankau, R. 1975. Prokaryote affinities of factors with the greatest effect on biocon- Duboscqia penetrans, Thorne. J. Protozool. 21:31-34. trol. Currently, no known computer pro- 16. Mankau, R. 1975. Bacillus penetrans n. comb. causing a virulent disease of plant-parasitic nema- grams simulate the influence of an antago- todes. J. Invertebr. Pathol. 26:333-339. nistic bacterium on a population of a plant- 17. Mankau, R., and J. L. Imbriani. 1975. The parasitic nematode. The computer tool life cycle of an endoparasite in some Tylenchid should improve our chances of success in nematodes. Nematologica 21:89-94. 18. Mankau, R., and R. J. Minteer. 1962. Reduc- biocontrol of plant-parasitic nematodes by tion of soil populations of the citrus nematode by allowing selective choice of the more in- the addition of organic materials. Plant Dis. Reptr. fluential factors and by reducing the num- 46:375-378. ber of tests required. 19. Mankau, R., and N. Prasad. 1972. Possibil- ities and problems in the use of a sporozoan endo- parasite for biological control of plant parasitic LITERATURE CITED nematodes. Nematropica 2:7. 20. Mankau, R., and N. Prasad. 1977. Infectivity 1. Adams, R. E., and J. J. Eichenmuller. 1963. of Bacilius penetrans in plant parasitic nematodes. A bacterial infection of Xiphinema americanum. J. Nematol. 9:40-45. Phytopathology 53:745 (Abstr.). 21. Metchnikoff, M. E. 1888. Pasteuria ramosa, 2. Allen, M. W. 1957. A new species of the un reprOsentant des bact6ries ~ division longi- genus Dolichodorus from California (Nematoda: tudinales. Ann. Inst. Pasteur Paris 2:165-170. Tylenchida). Proc. Helm. Soc. Wash. 24:95-98. 22. Odum, E. P., and H. T. Odum. 1959. Funda- 3. Canning, E. U. 1973. Protozoal parasites as mentals of ecology. Philadelphia, Saunders, 546 pp. agents for biological control of plant-parasitic 23. Perry, J. N. 1978. A population model for nematodes. Nematologica 19:342-348. the effect of parasitic fungi on numbers of the cereal 4. Cross, T. 1970. The diversity of bacterial cyst nematode Heterodera avenae. J. Appl. Ecol. spores. J. Appl. Bact. 33:95-102. 15:781-788. 5. Dutky, E. M. 1978. Some factors affecting in- 24. Rodriguez-Kabana, R., J. w. Jordan, and fection of plant-parasitic nematodes by a bacterial J. P. Hollis. 1965. Nematodes: biological control in spore parasite of nematodes. M.S. Thesis, Dept. of rice fields: role of hydrogen sulfide. Science 148:524- Botany, University of Maryland, 44 p. 526. 6. Dutky, S. R. 1963. The milky diseases. In: 25. Sayre, R. M., and W. P. Wergin. 1977. Bac- 270 Journal of Nematology, Volume 12, No. 4, October 1980 terial parasite of a plant nematode: morphology and Pasteuria-Blastobacter group. Can. J. Microbiol. 19: ultrastructure. J. Bacteriol. 129:1091-1101. 609-614. 26. Sayre, R. M., W. P. Wergin, and R. E. Davis. 30. Thorne, G. 1940. Duboscqia penetrans n. sp. 1977, Occurrence of Moina rectirostris (: (Sporozoa, Microsporidia, Nosematidae), a parasite Daphnidae) of a plant morphologically similar to of the nematode Pratylenchus pratensis (de Man) Pasteuria ramosa (Metchnikoff, 1888). Can. J. Filipjev. Proc. Helm. Soc. Wash. 7:52-53. Microbiol. 23:1573-1579. 31. Walker, J. T. 1969. Pratylenchus penetrans 27. Shepherd, A. M., S. A. Clark, and A. Kemp- (Cobb) populations as influenced by microorganisms ton. 1973. An intracellular microorganism associated and soil amendments. J. Nematol. 1:260-264. with tissues of Heterodera spp. Nematologica 19: 32. Williams, J. R. 1960. Studies on the nema- 31-34. tode soil fauna of sugarcane fields in Mauritius. 28. Slack, J. M., and M. A. Gerencser. 1975. Nematologica 5:37-42. Actinomyces, filamentous bacteria; biology and 33. Williams, J. R. 1967. Observations on para- pathogenicity. Burgess Publishing Co., Minneapolis. sitic protozoa in plant-parasitic and free-living nem- p. 175. atodes. Nematologica 13:336-342. 29. Staley, J. T. 1973. Budding bacteria of the

Biocontroh The Potential of Entomophilic Nematodes in Insect Management 1 John M. Webster 2 Abstract: A review of the development of entomophilic nematology and a commentary on the potential of entomophilic nematodes in controlling insect pests. The paper considers some of the major contributions to our knowledge of entomophilic nemagology; factors involved in insect pest management and how they are applicable to the use of nematodes; nematodes which are most promising as biological control agents; and problems to be solved to facilitate the use of entomophilic nematodes in insect management. Key Words: Mermis nigrescens, Neoaplectana spp., Romanomermis culicivorax, Deladenus siricidicola, Tetradonema plicans.

"A modern endeavour is to bring insect realized. Why? In the following discussion pests under natural control ..... The I will give some of the reasons. 'natural control' of insects will be most effective if all possible agencies and fac- MILEPOSTS tors are utilized; among these agencies nemas are by no means negligible." Mermis nigrescens, though atypical of the family, is often considered the type or- This is a quotation from Nathan Cobb ganism for entomophilic hematology and (12) in the 1927 USDA Yearbook of Agri- was described by Dujardin in France 137 culture. In recent years, pest management years ago (16). About 20 years later, Sir philosophy has developed in ways that John Lubbock, in Edinburgh (34), pro- avoid harm to useful organisms. As nema- vided biological and taxonomic informa- todes that kill pest insects are usually harm- tion on Sphaerularia bombi that widened less to other organisms, they have enormous contemporary perspectives on the nature of potential for use in place of chemical pesti- nematode-insect associations. Numerous cides in pest management systems. Never- publications in entomophilic nematology theless, more than 50 years after Cobb's followed, especially in Germany, and this statement, this potential has yet to be fully was crowned in the early part of the 20th century by a detailed taxonomic account of Received for publication 18 February 1980. the mermithids by Hagmeier (27) of the 1Symposium paper presented at the annual meeting of University of Heidelberg. Cobb (10) entered the Society of Nematologists, Salt Lake City, Utah, U.S.A., 23-26 July 1979. the arena in 1920 with descriptions of sev- ~Pestology Centre, Department of Biologicat Sciences, eral new species of entomophilic nematodes Simon Fraser University, Burnaby, Vancouver, Canada. V5A ] $6. and followed by his famous observations on I thank Dr. B. P. Beirne of Simon Fraser University the biology and of mermithids and Dr. J. J. Petersen of the United States Department of Agriculture for comments on the manuscript. (11,13). During the first half of the 20th cen-