Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

Antibodies from Chicken Eggs as Probes for Antigens from Pasteuria penetrans Endospores

The bacteria Pasteuria spp. have been identified as among the most promising of several microbial organisms currently under investigation as biological control agents of plant-parasitic nematodes. As part of our goal to develop methods to discriminate isolates of Pasteuria penetrans with different host preferences, we investigated the potential of developing antibody probes to identify endospores of different isolates of P. penetrans. Polyclonal IgY antibodies were raised in chickens against endospores of P. penetrans isolates P20 and P100. Hens were injected with P20 or P100 endospore suspensions and boosted at 14 days. Anti-spore titers were determined with ELISA on yolk extracts of individual eggs as a function of time. The highest titers were found in eggs produced at 22 to 35 days after initial injections. Yolk extracts showing the highest titers were combined and processed to provide partially purified IgY preparations. SDS-PAGE and immunoblot analyses identified protein antigens with Mr values of 23-24, 46, and 57-59 KDa common to both P20 and P100 endospores. One protein antigen with an Mr value of 62 KDa was unique to the PI00 endospores. The IgY antibodies reduced the attachment of Pasteuria endospores to their nematode hosts, indicating antibody interaction with antigens on the endospore surface that are involved in the recognition and attachment processes.     

Phylogenetic Analysis of Pasteuria penetrans by 16S rRNA Gene Cloning and Sequencing

Pasteuria penetrans is an endospore-forming bacterial parasite of Meloidogyne spp. This organism is among the most promising agents for the biological control of root-knot nematodes. In order to establish the phylogenetic position of this species relative to other endospore-forming bacteria, the 16S ribosomal genes from two isolates of P. penetrans, P-20, which preferentially infects M. arenaria race 1, and P-100, which preferentially infects M. incognita and M. javanica, were PCR-amplified from a purified endospore extraction. Universal primers for the 16S rRNA gene were used to amplify DNA which was cloned, and a nucleotide sequence was obtained for 92% of the gene (1,390 base pairs) encoding the 16S rDNA from each isolate. Comparison of both isolates showed identical sequences that were compared to 16S rDNA sequences of 30 other endospore-forming bacteria obtained from GenBank. Parsimony analyses indicated that P. penetrans is a species within a clade that includes Alicyclobacillus acidocaldarius, A. cycloheptanicus, Sulfobacillus sp., Bacillus tusciae, B. schlegelii, and P. ramosa. Its closest neighbor is P. ramosa, a parasite of Daphnia spp. (water fleas). This study provided a genomic basis for the relationship of species assigned to the genus Pasteuria, and for comparison of species that are parasites of different phytopathogenic nematodes.     

Genetic and Immunological Comparison of the Cladoceran Parasite Pasteuria ramosa with the Nematode Parasite Pasteuria penetrans

Pasteuria penetrans, an obligate endospore-forming parasite of Meloidogyne spp. (root knot nematodes), has been identified as a promising agent for biocontrol of these destructive agricultural crop pests. Pasteuria ramosa, an obligate parasite of water fleas (Daphnia spp.), has been shown to modulate cladoceran populations in natural ecosystems. Selected sporulation genes and an epitope associated with the spore envelope of these related species were compared. The sigE and spoIIAA/spoIIAB genes differentiate the two species to a greater extent than 16S rRNA and may serve as probes to differentiate the species. Single-nucleotide variations were observed in several conserved genes of five distinct populations of P. ramosa, and while most of these variations are silent single-nucleotide polymorphisms, a few result in conservative amino acid substitutions. A monoclonal antibody directed against an adhesin epitope present on P. penetrans P20 endospores, previously determined to be specific for Pasteuria spp. associated with several phytopathogenic nematodes, also detects an epitope associated with P. ramosa endospores. Immunoblotting provided patterns that differentiate P. ramosa from other Pasteuria spp. This monoclonal antibody thus provides a probe with which to detect and discriminate endospores of different Pasteuria spp. The presence of a shared adhesin epitope in two species with such ecologically distant hosts suggests that there is an ancient and ecologically significant recognition process in these endospore-forming bacilli that contributes to the virulence of both species in their respective hosts.     

A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies

A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.     

Pasteuria penetrans for Control of Meloidogyne incognita on Tomato and Cucumber,and M. arenaria on Snapdragon

Meloidogyne incognita and Meloidogyne arenaria are important parasitic nematodes of vegetable and ornamental crops. Microplot and greenhouse experiments were conducted to test commercial formulations of the biocontrol agent Pasteuria penetrans for control of M. incognita on tomato and cucumber and M. arenaria on snapdragon. Three methods of application for P. penetrans were assessed including seed, transplant, and post-plant treatments. Efficacy in controlling galling and reproduction of the two root-knot nematode species was evaluated. Seed treatment application was assessed only for M. incognita on cucumber. Pasteuria treatment rates of a granular transplant formulation ranged from 1.5 × 10⁵ endospores/cm³ to 3 × 10⁵ endospores/cm³ of transplant mix applied at seeding. Additional applications of 1.5 × 10⁵ endospores/cm³ of soil were applied as a liquid formulation to soil post-transplant for both greenhouse and microplot trials. In greenhouse cucumber trials, all Pasteuria treatments were equivalent to steamed soil for reducing M. incognita populations in roots and soil, and reducing nematode reproduction and galling. In cucumber microplot trials there were no differences among treatments for M. incognita populations in roots or soil, eggs/g root, or root condition ratings. Nematode reproduction on cucumber was low with Telone II and with the seed treatment plus post-plant application of Pasteuria, which had the lowest nematode reproduction. However, galling for all Pasteuria treatments was higher than galling with Telone II. Root-knot nematode control with Pasteuria in greenhouse and microplot trials varied on tomato and snapdragon. Positive results were achieved for control of M. incognita with the seed treatment application on cucumber.     

Host Suitability of the Olive Cultivars Arbequina and Picual for Plant-Parasitic Nematodes

Host suitability of olive cultivars Arbequina and Picual to several plant-parasitic nematodes was studied under controlled conditions. Arbequina and Picual were not suitable hosts for the root-lesion nematodes Pratylenchus fallax, P. thornei, and Zygotylenchus guevarai. However, the ring nematode Mesocriconema xenoplax and the spiral nematodes Helicotylenchus digonicus and H. pseudorobustus reproduced on both olive cultivars. The potential of Meloidogyne arenaria race 2, M. incognita race 1, and M. javanica, as well as P. vulnus and P. penetrans to damage olive cultivars, was also assessed. Picual planting stocks infected by root-knot nematodes showed a distinct yellowing affecting the uppermost leaves, followed by a partial defoliation. Symptoms were more severe on M. arenaria and M. javanica-infected plants than on M. incognita-infected plants. Inoculation of plants with 15,000 eggs + second-stage juveniles/pot of these Meloidogyne spp. suppressed the main height of shoot and number of nodes of Arbequina, but not Picual. Infection by each of the two lesion nematodes (5,000 nematodes/pot) or by each of the three Meloidogyne spp. suppressed (P < 0.05) the main stem diameter of both cultivars. On Arbequina, the reproduction rate of Meloidogyne spp. was higher (P < 0.05) than that of Pratylenchus spp.; on Picual, Pratylenchus spp. reproduction was higher (P < 0.05) than that of Meloidogyne spp.     

Host Range and Ecology of Isolates of Pasteuria spp. from the Southeastern United States

Isolates of Pasteuria penetrans were evaluated for ecological characteristics that are important in determining their potential as biological control agents. Isolate P-20 survived without loss of its ability to attach to its host nematode in dry, moist, and wet soil and in soil wetted and dried repeatedly for 6 weeks. Some spores moved 6.4 cm (the maximum distance tested) downward in soil within 3 days with percolating water. The isolates varied greatly in their attachment to different nematode species and genera. Of five isolates tested in spore-infested soil, three (P-104, P-122, B-3) attached to two or more nematode species, whereas B-8 attached only to Meloidogyne hapla and B-I did not attach to any of the nematodes tested. In water suspensions, spores of isolate P-20 attached readily to M. arenaria but only a few spores attached to other Meloidogyne spp. Isolate P-104 attached to all Meloidogyne spp. tested but not to Pratylenchus scribneri. Isolate B-4 attached to all species of Meloidogyne and Pratylenchus tested, but the rate of attachment was relatively low. Isolate P-Z00 attached in high numbers to M. arenaria when spores were extracted from females of this nematode; when extracted from M. javanica females, fewer spores attached to M. arenaria than to M. javanica or M. incognita.     

Pasteuria sp. Parasitizing Trophonema okamotoi in Florida

Two populations of Trophonema okamotoi parasitized by Pasteuria sp. were found on Liquidambar styraciflua (sweetgum) and on an unidentified tropical grass in north-central Florida. Endospores of this Pasteuria sp. attached to motile vermiform second-stage juveniles (J2) and males of T. okamotoi, but not to other developmental stages. Sporangia and new endospores were produced only inside the bodies of swollen and sedentary third- and fourth-stage juveniles and females that developed in the host roots. No egg masses were produced by infected T. okamotoi females. The endospore diameter from the tropical grass population was 4.93 μm and the central core diameter was 1.97 μm; measurements of endospores from the sweetgum populations were similar. Endospores that were collected from T. okamotoi and added to uninfected T. okamotoi and other plant-parasitic nematodes attached/to J2 of T. okamotoi but did not attach to juveniles and adults of Helicotylenchus pseudorotrustus, Pratylenchus brachyurus, or to J2 of either Meloidogyne arenaria race 1, M. incognita race 1, M. javanica, or Tylenchulus semipenetrans. Pasteuria sp. from T. okamotoi differed from the described Pasteuria species in endospore size, host preference, and rate of attachment.     

Extraction and Purification of Pasteuria spp. Endospores

Pasteuria penetrans is an endospore-forming bacterial parasite of root-knot nematodes that has potential as a biological control agent. Biochemical investigations of P. penetrans are limited because of difficulty in obtaining large quantities of endospores free of plant debris and contaminating microorganisms. Our objective was to develop a technique for extraction and purification of P. penetrans endospores from root-knot nematodes. Tomato roots infected with Meloidogyne arenaria that was parasitized by P. penetrans were digested with cytolase. The nematode females along with plant debris were washed with a jet stream of water onto an 800-µm-pore sieve nested on a 250-µm-pore sieve. The materials retained on the 250-µm-pore sieve were centrifuged through a 20% sucrose solution. The resulting loose pellet fraction was collected on a 250-µm-pore sieve and then centrifuged through a 47% sucrose solution. Endospore-filled females were handpicked from the 47% sucrose pellicle fraction. Endospores were released by grinding the females with a glass tissue grinder. The endospores were then filtered through a nylon filter with 8-µm openings, collected by centrifugation, and subjected to buoyant density centrifugation in different media. Further purification by buoyant density centrifugation in a linear gradient of sodium diatrizoate resulted in a preparation of endospores free of debris. This additional step may be desirable for the further characterization of components unique to the endospores.     

Nematode Community Structure in a Vineyard Soil

Distribution of the nematode community in a California vineyard was studied over a 13-month period. Omnivorous and microbivorous nematodes were similarly distributed in the root zone, with greatest densities occurring between vine rows and near the soil surface. Greatest densities of plant-parasitic nematodes were found in the vine row, with the individual species differing in their vertical distribution. Total nematode biomass was greatest between rows near the surface. Biomass of plant parasites was greatest in the upper 30 cm of soil in the row, whereas biomass of microbivores was greatest in this region between rows. Of the plant-parasitic nematodes, the variability in distribution among vines was greatest for Paratylenchus hamatus and least for Meloidogyne spp.     

Occurrence of Pasteuria-like Organisms on Selected Plant-Pamsitic Nematodes of Pineapple in the Hawaiian Islands

Soils from 320 sites representing diverse undisturbed habitats from five Hawaiian Islands were assessed for occurrence of Pasteuria-like organisms. Mean annual rainfall at sites ranged from 125-350 cm, elevation from 69-2,286 m, and annual mean temperature from 12-24 C. Seven different natural communities were represented: wet lowland, mesic lowland, wet montane, mesk montane, dry montane, mesic subalpine, and dry alpine. Pasteuria spp. in a soil sample was detected by baiting with infective stages of Helicotylenchus dihystera, Meloidogyne javanica, Pratylenchus brachyurus, and Rotylenchulus reniformis, followed by cultivation of the nematodes on pineapple plants for 10-11 months. All nematode baits except R. reniformis were readily recovered from the soil samples. A sample was considered Pasteuria-positive if at least 5 % of the nematode specimens showed endospore attachment. Thirteen percent of all samples were positive for Pasteuria-like organisms. The frequencies of association between Pasteuria spp. and Meloidogyne, Helicotylenchus, or Pratylenchus species were 52%, 24%, and 24%, respectively. Positive samples were more prevalent on the older islands of Kauai and Oahu (75%), in lowland communities (61%), and in areas with introduced vegetation (60%). More than 27% of the positive samples were associated with plant species in a few selected families that included Meliaceae and Myrtaceae. Occurrence of Pasteuria spp. seemed to be positively associated with mean annual rainfall or temperature, but negatively associated with elevation.     

Temperature-Dependent Development of Pasteuria penetrans in Meloidogyne arenaria

Pasteuria penetrans is a promising biological control agent of plant-parasitic nematodes. This study was conducted to determine effects of temperature on the bacterium''s development in Meloidogyne arenaria. Developmental stages of P. penetrans were viewed with a compound microscope and verified with scanning electron microscopy within each nematode at 100 accumulated degree-day intervals by tracking accumulated degree-days at three temperatures (21, 28, and 35 °C). Five predominant developmental stages of P. penetrans were identified with light microscopy: endospore germination, vegetative growth, differentiation, sporulation, and maturation. Mature endospores were detected at 28, 35, and >90 calendar days at 35, 28, and 21 °C, respectively. The number of accumulated degree-days required for P. penetrans to reach a specific developmental stage was different for each temperature. Differences were observed in the development of P. penetrans at 21, 28, and 35 °C based on regression values fitted for data from 100 to 600 accumulated degree-days. A linear response was observed between 100 to 600 accumulated degree-days; however, after 600 accumulated degree-days the rate of development of P. penetrans leveled off at 21 and 28 °C, whereas at 35 °C the rate decreased. Results suggest that accumulated degree-days may be useful only in predicting early-developmental stages of P. penetrans.     

Axenizing and Culturing Endomigratory Plant-Parasitic Nematodes using Pluronic F127, Including its Effects,on Population Dynamics of Pratylenchus penetrans

A non-chemical technique for surface sterilizing plant-parasitic nematodes for aseptic cultures is described. The method is most applicable to nematodes with active migratory infective stages and requires only a few starting specimens. Rate of achieving a primary aseptic culture with the technique ranged from 60%-100% depending on the conditions of the specimens collected for culturing. Aseptic cultures of species of Meloidogyne, Rotylenchuluz, Pratylenchus, and Radopholus initiated with the method remained contamination-free after 12 months of maintenance in tomato root explant or alfalfa callus cultures. Further studies of Pluronic F127, a polyol gel medium employed in the technique to confine the spread of contaminating bacteria or fungi associated with the nematodes, showed that the polyol gel was a suitable support medium for culturing corn root explant, alfalfa callus tissues, and consequently Pratylenchus species including P. agilis, P. brachyurus, P. scribneri, and P. penetrans. During the course of 10 months, P. penetrans reared in polyol-base medium followed a standard biological growth curve, multiplied to a higher population density, maintained a similar female-to-male ratio, and possessed a similar tendency to reside inside or outside host tissues as did P. penetrans reared in agar-base medium. The percentages of P. penetrans juveniles in the sub-populations residing outside or inside the host tissues reared in polyol-base medium also were similar to and fluctuated temporally in like manner as those reared in agar-base medium. Members of these sub-populations from the polyol- or agar-base were equally infective and reproductive after 9 months of culturing.     

Temperature Effects on the Attachment of Pasteuria penetrans Endospores to Meloidogyne arenaria Race 1

Pasteuria penetrans is a gram positive bacterium that prevents Meloidogyne spp. from reproducing and diminishes their ability to penetrate roots. The attachment of the endospores to the cuticle of the nematodes is the first step in the life cycle of the bacterium and is essential for its reproduction. As a preliminary study to a field solarization test, the effects of temperature on the attachment of P. penetrans on Meloidogyne arenaria race 1 were investigated. Preexposing second-stage juveniles (J2) of M. arenaria to approximately 30 °C in water before exposing them to endospores increased their receptivity to endospore attachment when compared to treating J2 at 25 °C or 35 °C. In tests with soil, highest attachment occurred when J2 were incubated in soil infested with endospores and maintained at 20 °C to 30 °C for 4 days. Heating J2 in soil to sublethal temperatures (35 °C to 40 °C) decreased endospore attachment. Incubating P. penetrans endospores in soil at 30 °C to 70 °C for 5 hours a day over 10 days resulted in reductions of endospore attachment to nematodes as temperatures of incubation increased to 50 °C and higher.     

Techniques for image analysis of movement of juveniles of root-knot nematodes encumbered with Pasteuria penetrans spores

Root-knot nematodes (Meloidogyne spp.) are the most significant plant-parasitic nematodes that damage many crops all over the world. The free-living second stage juvenile (J2) is the infective stage that enters plants. The J2s move in the soil water films to reach the root zone. The bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes, is cosmopolitan, frequently encountered in many climates and environmental conditions and is considered promising for the control of Meloidogyne spp. The infection potential of P. penetrans to nematodes is well studied but not the attachment effects on the movement of root-knot nematode juveniles, image analysis techniques were used to characterize movement of individual juveniles with or without P. penetrans spores attached to their cuticles. Methods include the study of nematode locomotion based on (a) the centroid body point, (b) shape analysis and (c) image stack analysis. All methods proved that individual J2s without P. penetrans spores attached have a sinusoidal forward movement compared with those encumbered with spores. From these separate analytical studies of encumbered and unencumbered nematodes, it was possible to demonstrate how the presence of P. penetrans spores on a nematode body disrupted the normal movement of the nematode.     

Molecular Diagnosis of Meloidogyne Species

Genetic variation within nuclear and mitochondrial DNA of Meloidogyne species and host races has been evaluated for the development of root-knot nematode molecular diagnostics. This review summarizes the distinctive features of several useful DNA-based assays for plant-parasitic nematodes, focusing upon the direct application of these procedures for Meloidogyne detection, identification, and systematics.     

Correlation of Edaphic Factors with Plant-parasitic Nematode Population Densities in a Forage Field

Two hundred soil samples from the A_p horizon of a reed canarygrass field overlaying several different but related soils in northern Minnesota were analyzed for plant-parasitic nematodes and 22 edaphic factors. Pratylenchus penetrans was the predominant nematode taxon. Others were Aglenchus agricola, Tylenchorhynchus spp., Heterodera trifolii, Paratylenchus spp., Tylenchus maius, and Criconemella sp. Five nematode taxa, P. penetrans, A, agricola, Tylenchorhynchus spp., H. trifolii, and Paratylenchus spp., were correlated with particle size, Tylenchus maius and Criconemella sp. were correlated with effective cation exchange capacity. Nematode field spatial arrangements were related to a combination of statistically significant positive and negative soil factor effects on the nematode populations. Contour maps derived by geostatistical techniques were used to visually validate statistically significant correlations of nematode and soil data. Contour mapping to supplement traditional statistical techniques can be used to achieve a more holistic approach to studies of nematode-soil interrelationships.     

Phenotypic Adaptations in Pasteuria spp. Nematode Parasites

The endospore and central core diameters of 69 isolates of Pasteuria spp. showed a relationship with the body wall thickness of their corresponding host nematodes. A relationship was also observed when cuticle and hypodermis layer data were derived from transmission electron microscopy micrographs. Principal component analysis and hierarchical cluster analysis based on endospore and central core diameters and host nematode body wall thickness delineated six distinct groups. Five groups included nematode species of distinct taxa. Endospore morphometric diversity appears to be the result of an evolutionary adaptation that occurred during host nematode speciation related to the forces acting on adhering endospores and(or) to the host cuticle penetration phase. On this basis, the validity of endospore morphometrics and host taxonomy as significant parameters in discriminating Pasteuria species is questioned.