Biocontrol: Bacillus 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 Pasteuria, 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 bacteria 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 • 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, Daphnia 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 • i FOIIMATION FOI~MATION VII OIETATIVIE FOR|SPORE OF OF ENDOSPORE GeOWTH SPORE CORTEX COATS