Suppression of Meloidogyne arenaria Race 1 by Soil Application of Endospores of Pasteuria penetrans

The potential of Pasteuria penetrans for suppressing Meloidogyne arenaria race 1 on peanut (Arachis hypogaea) was tested over a 2-year period in a field microplot experiment. Endospores of P. penetrans were mass-produced on M. arenaria race 1 infecting tomato plants. Endospores were inoculated in the first year only at rates of 0, 1,000, 3,000, 10,000, and 100,000 endospores/g of soil, respectively, into the top 20 cm of microplots that were previously infested with M. arenaria race 1. One peanut seedling was planted in each microplot. In the first year, root gall indices and pod galls per microplot were significantly reduced by 60% and 95% for 100,000 endospores/g of soil, and 20% and 65% for 10,000 endospores/g of soil, respectively. Final densities of second-stage juveniles (J2) in soil were not significantly different among the treatments. The number of endospores attached to J2 and percentage of J2 with attached endospores significantly increased with increasing endospore inoculation levels. Pasteuria penetrans significantly reduced the densities of J2 that overwintered. In the second year, root and pod gall indices, respectively, were significantly reduced by 81% and 90% for 100,000 endospores/g of soil, and by 61% and 82% of 10,000 endospores/g of soil. Pod yields were significantly increased by 94% for 100,000 and by 57% for 10,000 endospores/g of soil, respectively. The effect of P. penetrans on final densities of J2 in soil was not significant. Regression analyses verified the role of P. penetrans in the suppression of M. arenaria. The minimum number of endospores required for significantly suppressing M. arenaria race 1 on peanut was 10,000 endospores/g of soil.     

Effects of Formulation and Host Nematode Density on the Ability of In Vitro-Produced Pasteuria Endospores to Control its Host Belonolaimus longicaudatus

The effect of nematode population density at the time of application and formulations of in vitro-produced Pasteuria spp. endospores on the final population density of Belonolaimus longicaudatus was studied in an 84-d-long pot bioassay. The experiment utilized a factorial design consisting of 30 or 300 B. longicaudatus /100 cm(3) of sandy soil and three formulations of in vitro-produced Pasteuria spp. endospores (nontreated, granular, or liquid). No differences were observed in percent endospore attachment between nematode inoculum levels during either trial. Granular and liquid formulations of in vitro-produced endospores suppressed nematode population densities by 22% and 59% in the first trial and 20% and 63% in the second, respectively compared with the nontreated control. The liquid formulation increased percent endospore attachment by 147% and 158%, respectively, compared with the granular formulation. The greatest root retention by the host plant was observed at the lower B. longicaudatus inoculation level following application of the liquid formulation. While both the granular and liquid formulations reduced B. longicaudatus population densities in the soil, the liquid spore suspension was most effective.     

Effects of Monoclonal Antibodies,Cationized Ferritin,and Other Organic Molecules on Adhesion of Pasteuria penetrans Endospores to Meloidogyne incognita

The incidence of adhesion of Pasteuria penetrans endospores to Meloidogyne incognita second-stage juveniles (J2) was studied after pretreatment of the latter with monoclonal antibodies (MAb), cationized ferritin, and other organic molecules in replicated trials. Monoclonal antibodies developed to a cuticular epitope of M. incognita second-stage juveniles gave significant reductions in attachment of P. penetrans endospores to treated nematodes. MAb bound to the entire length of J2 except for the area of the lateral field, where binding was restricted to the incisures. Since reductions in attachment with MAb treatment were modest, it is uncertain if these results implicated a specific surface protein as a factor that interacted in binding of the endospore to the nematode cuticle. Endospore attachment was decreased following treatment of the nematode with the detergents sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). Endospore attachment to live nematodes was significantly greater than attachment to dead nematodes. Attachment rates of three P. penetrans isolates to M. incognita race 3 varied between isolates. The effects of neuraminidase, pronase, pepsin, trypsin, lipase, and Na periodate on endospore attachment were inconsistent. The cationic dye alcian blue, which binds sulfate and carboxyl groups on acidic glycans, had no consistent effect on endospore attachment. The incidence of endospore attachment was significantly lower but modest, at best, for nematodes that were treated with cationized ferritin alone or cationized ferritin following monoclonal antibody. The lack of consistency or extreme reduction in most experiments suggests that attachment of P. penetrans spores to M. incognita is not specified by only one physico-chemical factor, but may involve a combination of at least two physico-chemical factors (including surface charge and movement of the J2). This points to a need for analysis of combined or factorial treatment effects.     

Population Dynamics of Meloidogyne incognita,M. arenaria,and Other Nematodes and Crop Yields in Rotations of Cotton,Peanut, and Wheat Under Minimum Tillage

Wheat, cotton, and peanut were arranged in three cropping sequences to determine the effects of fenamiphos (6.7 kg a.i./ha) and cropping sequence on nematode population densities and crop yields under conservation tillage and irrigation for 6 years. The cropping sequences included a wheat winter cover crop each year and summer crops of cotton every year, peanut every year, or cotton rotated every other year with peanut. The population densities of Meloidogyne spp. and Helicotylenchus dihystera were determined monthly during the experiment. Numbers of M. incognita increased on cotton and decreased on peanut, whereas M. arenaria increased on peanut, and decreased on cotton; both nematode species remained in moderate to high numbers in plots of wheat. Root damage was more severe on cotton than peanut and was not affected by fenamiphos treatment. The H. dihystera population densities were highest in plots with cotton every summer, intermediate in the cotton-peanut rotation, and lowest in plots with peanut every summer. Over all years and cropping sequences, yield increases in fenamiphos treatment over untreated control were 9% for wheat, 8% for cotton, and 0% for peanut. Peanut yields following cotton were generally higher than yields following peanut. These results show that nematode problems may be manageable in cotton and peanut production under conservation tillage and irrigation in the southeastern United States.     

Evaluation of an Antibiotic-Producing Strain of Pseudomonas fluorescens for Suppression of Plant-Parasitic Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG), produced by some strains of Pseudomonas spp., is involved in suppression of several fungal root pathogens as well as plant-parasitic nematodes. The primary objective of this study was to determine whether Wood1R, a D-genotype strain of DAPG-producing P. fluorescens, suppresses numbers of both sedentary and migratory plant-parasitic nematodes. An experiment was conducted in steam-heated soil and included two seed treatments (with Wood1R and a control without the bacterium) and six plant-nematode combinations which were Meloidogyne incognita on cotton, corn, and soybean; M. arenaria on peanut; Heterodera glycines on soybean; and Paratrichodorus minor on corn. Wood 1R had no effect on final numbers of M. arenaria, P. minor, or H. glycines; however, final numbers of M. incognita were lower when seeds were treated with Wood1R than left untreated, and this reduction was consistent among host plants. Population densities of Wood1R were greater on the roots of corn than on the other crops, and the bacterium was most effective in suppressing M. incognita on corn, with an average reduction of 41%. Despite high population densities of Wood1R on corn, the bacterium was not able to suppress numbers of P. minor. When comparing the suppression of M. incognita on corn in natural and steam-heated soil, egg production by the nematode was suppressed in natural compared to steamed soil, but the presence of Wood1R did not result in additional suppression of the nematodes in the natural soil. These data indicate that P. fluorescens strain Wood1R has the capacity to inhibit some populations of plant-parasitic nematodes. However, consistent suppression of nematodes in natural soils seems unlikely.     

Endospore heterogeneity in Pasteuria penetrans related to adhesion to plant-parasitic nematodes

Hybridoma cell lines were screened by indirect immunofluorescence for the secretion of monoclonal antibodies (Mabs) to the surface of a population of endospores of the obligate nematode hyperparasite Pasteuria penetrans. Whereas polyclonal antibodies from test sera samples recognized 100% of the spores, five selected Mabs recognized different proportions of the spores ranging from 10 to 90% showing that the spore population was highly heterogeneous. Probing spores adhering to different nematode populations with the five Mabs showed that different subpopulations of the spores were specific to different nematode populations indicating cuticular heterogeneity among the nematode populations. Each of the five Mabs recognized a far larger proportion of the spores adhering to the nematode population on which the Pasteuria population was originally cultured than of those adhering to other populations.     

Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing arctic ice cores

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% +/- 3.8% and 48.9% +/- 4.5%, respectively), while only 27.8% +/- 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 +/- 19 germinable spores/ml and 369 +/- 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% +/- 9.3%), and the second core contained 131 +/- 4 germinable spores/ml and 162 +/- 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% +/- 8.8%), whereas only 2 CFU/ml were detected by culturing.     

Crop Rotation and Races of Meloidogyne incognita in Cotton Root-knot Management

The influence o f various crop rotations and nematode inoculum levels on subsequent population densities of Meloidogyne incognita races 1 and 3 were studied in microplots. Ten different 3-year sequences o f cotton, corn, peanut, or soybean, all with cotton as the 3rd-year crop, were grown in microplots infested with each race. Cotton monoculture, two seasons o f corn, or cotton followed by corn resulted in high race 3 population densities and severe root galling on cotton the 3rd year. Peanut for 2 years preceding cotton most effectively decreased the race 3 population and root galls on cotton the 3rd year. Race 1 did not significantly influence cotton growth or yield at initial populations of up to 5,000 eggs/500 cm³ soil. At 5,000 eggs/500 cm³, cotton growth was suppressed by race 3 but yield was not affected.     

Parasitism of Helicotylenchus lobus by Pasteuria penetrans in Naturally Infested Soil

The population density of Helicotylenchus lobus and the percentage of the population with spores of Pasteuria penetrans were determined for 10 monthly intervals in naturally infested turf grass soil at Riverside, California. The percentage of nematodes with attached spores ranged from 40% to 67%. No relationship was found between nematode density and the percentage of nematodes with spores. The mean and maximum numbers of spores adhering per nematode with at least one spore ranged from 2 to 8 and 7 to 66, respectively. The mean number of spores per nematode (based on total number of H. lobus) was correlated with the percentage of nematodes with spores. Spores adhered to both adult and juvenile H. lobus. Between 9% and 32% of the nematodes with spores had been penetrated and infected by the bacterium. Many infected nematodes were dead, but mature spores were also observed within living adult and juvenile H. lobus that exhibited no apparent reduction in viability and motility. Spore and central endospore diameters of this P. penetrans isolate were larger than those reported for the type isolate from Meloidogyne incognita, but transmission and scanning electron microscopy did not reveal significant morphological differences between the two isolates. Spores of the isolate associated with H. lobus did not adhere to juveniles of M. incognita.     

Biological control potential of the obligate parasite Pasteuria penetransagainst the root-knot nematode, Meloidogyne incognita infestation in Brinjal

The efficacy of the obligate bacterial parasite, Pasteuria penetrans against the rootknot nematode, Meloidogyne incognita infestation was assessed in brinjal. The seedling pans with sterilized soil were inoculated with nematodes and root powder of P. penetrans were applied at different dosages viz., 0 x 10(6), 0.5 x 10(6) spores and 1 x 10(6) spores/pan. Seeds of brinjal cv Co2 were sown in the pans and seedlings were allowed to grow. The seedlings were transplanted to microplots containing sterilized soil. Observations on nematode infestation and plant growth were recorded at seedling, flowering, and fruiting stages. Nematode infestation was significantly reduced by P. penetrans treatment. There was 22, 75 and 86% reduction in nematode population of soil over control at seedling, flowering and fruiting stages, respectively, at higher spore density (1 x 10(6)). Egg mass production was decreased by 63, 78 and 89% over control at 35 (seedling), 100 (flowering) and 160 (fruiting) days after sowing respectively, at 1 x 10(6) spores treated soil. The parasitizing ability of P. penetrans increased with the age of the crop. At higher spore density the percentage of parasitization was increased from 52.0 (35 days after sowing) to 90.0 (160 days after sowing) %. At these stages of the crop, the spore load per juvenile also increased at the higher dose. The P. penetrans application enhanced the plant growth. The weight of the shoot was increased by 17.6% whereas root weight by 41.0% over the control at fruiting stage. The experimental results revealed the potential use of P. penetrans as biological control agent of M. incognita. Application of P. penetrans spores in the nursery is a good strategy since the mass multiplication is quite difficult.     

Distribution and Downward Movement of Pasteuria penetrans in Field Soil

Endospores of Pasteuria penetrans were evaluated for their vertical distribution in field soil and their downward movement through soil in the laboratory. In the field trial, the number of endospores attached to second-stage juveniles (J2) of Meloidogyne arenaria race 1 varied greatly in different soil depths. There were higher percentages of J2 with endospores attached in former weed fallow plots during the first 3 years of growing peanut than in former bahiagrass and rhizomal peanut plots (P ≤ 0.05). In weed fallow plots a higher average number of endospores per J2 were maintained in all depths, upper three depths, and upper four depths in 1999, 2000, and 2001, respectively (P ≤ 0.05). However, in 2002, there were no differences in the percentages of J2 with endospores attached and in the average of the numbers of endospores per J2 among the treatments (P > 0.05). In laboratory trials, P. penetrans endospores were observed to move throughout the soil through the percolation of water. After one application of water, some endospores were detected 25 to 37.5 cm deep. Endospores were present at the greatest depth, 37.5 to 50 cm, after the third application of water. These results indicate that rain or water applications by irrigation are likely to move endospores to deeper levels of the soil, but the majority of endospores remain in the upper 0-to-30-cm depth.     

Response of Peanut,Corn, Tobacco,and Soybean to Criconemella ornate

The relative susceptibility of four field crops to Criconemella ornata differed greatly in microplot tests. As few as 178 freshly-introduced C. ornata/500 cm³ of soil stunted peanut. In contrast, this nematode had no effect on the growth of corn or soybean. Large populations remaining after culture of peanut or corn enhanced the growth of tobacco. A problem of comparing the effects of a freshly introduced population of this nematode with large residual populations was encountered. Freshly extracted, greenhouse-grown inoculum caused the typical "yellows disease" on peanut, whereas much greater residual population densities following a poor host (tobacco) had little effect on the growth of peanut. It is suggested that many of the nematodes in the field following a poor host are dead. Peanut supported greater reproduction (up to 970-fold) than did other crops tested. Corn was intermediate, with a population increase as great as 264-fold; soybean and tobacco failed to maintain initial population densities.     

Effects of Crop Rotation and Nonfumigant Nematicides on Peanut and Corn Yields in Fields Infested with Criconemella Species

The effects of nematicide treatments and corn-peanut cropping sequences on the population development of Criconemella ornata, and C. sphaerocephala and the related impact on crop yields were investigated at two North Carolina locations. Criconemella ornata and C. sphaerocephala were present at the Norman Perry farm, Bertie County (BERTIE); however, only C. ornata was found at the Central Crops Research Station, Johnston County (CCRS). An untreated control was compared to aldicarb 15G, carbofuran 15G, ethoprop 10G, and terbufos 15G granular formulations applied at a rate of 2.2 kg a.i./ha. The cropping sequences were monocuhured corn (C-C-C); monocultured peanut (P-P-P); and two corn-peanut (C-P-C; P-C-P) rotations. Nematicides were inconsistent in controlling C. sphaerocephala and C. ornata. Nematicide treatments enhanced corn yields in the monoculture-cropping cycle in the final year of the experiment at CCRS. Peanut yields were greater in the rotated cropping sequence than under monoculture at BERTIE, but rotation had less effect on peanut yields at CCRS. Declining yields were correlated with an increase in numbers of nematodes. Corn was an intermediate host for C. sphaerocephala and a moderate to poor host for C. ornata. Peanut was an excellent host for C. ornata and a poor host for C. sphaerocephala.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Microbial imprinted polypyrrole/poly(3-methylthiophene) composite films for the detection of Bacillus endospores

The fabrication of Bacillus subtilis endospore imprinted conducting polymer films and subsequent electrochemical detection of bound spores is reported. Imprinted films were prepared by absorbing spores on the surface of glassy carbon electrodes upon which a polypyrrole, followed by a poly(3-methylthiophene), layer were electrochemically deposited. Spore template release was achieved through soaking the modified electrode in DMSO. Binding of endospores to imprinted films could be detected via impedance spectroscopy by monitoring changes in Y' (susceptance) using Mn(II)Cl2 (0.5M pH 3) as the supporting electrolyte. Here, the change in Y' could be correlated to spore densities between 10(4) and 10(7)cfu/ml. More sensitive detection of absorbed spores was achieved by following endospore germination via changes in film charge as measured using cyclic voltammetry. Here, imprinted films were submerged in spore suspensions to permit absorption, heat activated at 70 degrees C for 10 min prior to transferring to an electrochemical cell containing germination activators. By using the assay format it was possible to detect 10(2)cfu/ml. The observed changes in film charge could be attributed to the interaction of the supporting conducting polymer with dipicolinic acid (DPA) and other constituents released from the core in the course of germination. In all cases, it was not possible to regenerate the imprinted films without losing electrode response. In summary, the study has provided proof-of-concept for fabricating microbial imprinted films using conducting polymers.     

Crop Rotation and Herbicide Effects on Population Densities of Plant-Parasitic Nematodes

The influence of herbicides and mono- and multicropping sequences on population densities of nematode species common in corn, cotton, peanut, and soybean fields in the southeastern United States was studied for 4 years. Each experimental plot was sampled at monthly intervals. The application of herbicides did not significantly affect nematode population densities. Meloidogyne incognita and Trichodorus christiei increased rapidly on corn and cotton, but were suppressed by peanut and soybean. More Pratylenchus spp. occurred on corn and soybean than on cotton and peanut. Criconemoides ornatus increased rapidly on corn and peanut, but was suppressed by cotton and soybean. Helicotylenchus dihystera was more numerous on cotton and soybean than on corn and peanut. Numbers of Xiphinema americanum remained low on all crops. The peanut sequence was the most effective monocrop system for suppressing most nematode species. Multi-crop systems, corn-peanut-cotton-soybean and cotton-soybean-corn-peanut, were equally effective in suppressing nematode densities.     

Quantification of viable endospores from a Greenland ice core

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release during l-alanine induced germination via terbium ion (Tb3+)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295+/-19 spores mL(-1) and 369+/-36 spores mL(-1), respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27+/-2 spores mL(-1). The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 x 10(3)+/-6.7 x 10(2) cells mL(-1). Culturing attempts yielded 2 CFU mL(-1) (4 degrees C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays.     

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.     

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.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.