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.     

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.     

Suppression of Meloidogyne incognita and M. javanica by Pasteuria penetrans in Field Soil

The role of Pasteuria penetrans in suppressing numbers of root-knot nematodes was investigated in a 7-year monocuhure of tobacco in a field naturally infested with a mixed population of Meloidogyne incognita race 1 and M. javanica. The suppressiveness of the soil was tested using four treatments: autoclaving (AC), microwaving (MW), air drying (DR), and untreated. The treated soil bioassays consisted of tobacco cv. Northrup King 326 (resistant to M. incognita but susceptible to M. javanica) and cv. Coker 371 Gold (susceptible to M. incognita and M. javanica) in pots inoculated with 0 or 2,000 second-stage juveniles of M. incognita race 1. Endospores of P. penetrans were killed by AC but were only slightly affected by MW, whereas most fungal propagules were destroyed or inhibited in both treatments. Root galls, egg masses, and numbers of eggs were fewer on Coker 371 Gold in MW, DR, and untreated soil than in AC-treated soil. There were fewer egg masses than root galls on both tobacco cultivars in MW, DR, and untreated soil than in the AC treatment. Because both Meloidogyne spp. were suppressed in MW soil (with few fungi present) as well as in DR and untreated soil, the reduction in root galling, as well as numbers of egg masses and eggs appeared to have resulted from infection of both nematode species by P. penetrans.     

Attachment of Pasteuria penetrans Endospores to the Surface of Meloidogyne javanica Second-stage Juveniles

Pasteuria penetrans spore adhesion to Meloidogyne javanica second-stage juveniles (J2) was examined following several different pretreatments of the latter. The detergents sodium dodecyl sulfate and Triton X-100, the carbohydrates fucose and α-methyl-D-mannoside, and the lectins concanavalin A and wheat germ agglutinin reduced spore attachment. Spores exposed to M. javanica surface coat (SC) extract exhibited decreased adherence to the J2 surface. Second-stage juveniles that had been treated with antibodies recognizing a 250-kDa antigen of J2 SC extract had fewer spores attached to their surfaces, as compared to nontreated J2, except in the head region. This inhibition pattern was similar to that of antibody-labelling on M. javanica J2 as observed by electron microscopy. It is suggested that several SC components, such as carbohydrate residues, carbohydrate-recognition domains, and a 250-kDa antigen, are involved in P. penetrans spore attachment to the surface of M. javanica.     

Transfer and Development of Pasteuria penetrans

Pasteuria penetrans isolate P-20 has been attributed as the cause of soil suppressiveness to peanut root-knot nematode in Florida. In this study, P. penetrans was transferred from a suppressive site to a new site and established by growing susceptible hosts to the peanut root-knot nematode during both summer and winter seasons. When two soil fumigants, 1,3-dichloropropene (1,3-D) and chloropicrin, were applied broadcast at the rate of 168 liters/ha and 263 kg/ha, respectively, the bacterium was not adversely affected by 1,3-D but was adversely affected by chloropicrin. In autumn 2005, after the harvest of the second peanut crop, the greatest number of J2 was recorded in the chloropicrin-treated plots, followed by the non-fumigated plots and 1,3-D-fumigated plots. The percentage J2 encumbered with endospores, endospores per J2 and percentage of P. penetrans-infected females were greatest in the non-fumigated plots, followed by 1,3-D- and chloropicrin-fumigated plots. This study demonstrates that P. penetrans can be transferred from a suppressive site to a new site and increased to suppressive densities against the peanut root-knot nematode.     

Biological Control of Meloidogyne incognita by Paecilomyces lilacinus and Pasteuria penetrans

The root-knot nematode Meloidogyne incognita was controlled more effectively and yields of host plants were greater when Paecilomyces lilacinus and Pasteuria penetrans were applied together in field microplots than when either was applied alone. Yields of winter vetch from microplots inoculated with the nematode and with both organisms were not statistically different from yields from uninoculated control plots.     

Estimating Incidence of Attachment of Pasteuria penetrans Endospores to Meloidogyne spp. with Tally Thresholds

Pasteuria penetrans has .been identified as an important biological control agent of root-knot nematodes. In this study the use of tally thresholds was evaluated for estimating P. penetrans endospore attachment to second-stage juveniles (J2) of Meloidogyne spp. A tally threshold (T) is defined as the maximum number of individuals in a sample unit that may be treated as absent based on binomial sampling. Three different data sets that originated from centrifugal bioassay, incubation bioassay, and field experiments were investigated. The data sets each contained 70, 33, and 111 estimates of the mean number of endospores attached per J2 (m), respectively. Empirical relationships between m and proportions of J2 with ≤T endospores attached (P_T) were developed using parameters from the linear regression of ln(m) on P_T (0 < P_T < 1): ln(m) = a + b P_T, T was set to 0, 1, 2, 3, 4, 5, 8, and 10 endospores/J2. The results indicated that the variances of linear equations tended to decrease with increasing T values for all three data sets. T values of 0, 1, 8, and 10 endospores/J2 for centrifugal bioassay and incubation bioassay, and of 0, 1, 2, and 3 endospores/J2 for field experiments were associated with an r² of >= 0.8. These T values were robust for estimating m from P_T, reducing the variability as well as the time and effort spent in estimating the mean number of endospores attached per J2.     

Effect of Temperature on Attachment,Development, and Interactions of Pasteuria penetrans on Meloidogyne incognita

The effect of temperature (10, 20, 25, 30, and 35 C) on attachment and development of Pasteuria penetrans on Meloidogyne arenaria race 1 was elevated in growth chambers. The greatest attachment rate of endospores of P. penetrans occurred on second-stage juveniles at 30 C. The bacterium developed more quickly within its host at 30 and 35 C than at 25 C or below. The development of the bacterium within the nematode female was divided into nine recognizable life stages, which ranged from early vegetative thalli to mature sporangia. Mature sporangium was the predominant life stage observed after 35, 40, 81, and 116 days at 35, 30, 25, and 20 C, respectively. The body width and length of M. arenaria females infected with P. penetrans were smaller initially than the same dimensions in uninfected females, but became considerably larger over time at 25, 30, and 35 C. This isolate of P. penetrans also parasitized and completed its life cycle in males of M. arenaria.     

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.     

Monoclonal Antibodies to Secretory Granules in Esophageal Glands of Meloidogyne Species

Monoclonal antibodies to secretory granules in the dorsal or subventral esophageal glands were generated by injecting BALB/c mice with immunogens from preparasitic second-stage juveniles (J2) of Meloidogyne incognita. Antibodies specific for secretory granules in the J2 subventral esophageal glands or the dorsal gland were identified by indirect immunofluorescence microscopy. Only antibodies that reacted with granules in the J2 dorsal gland reacted with the esophageal gland lobe ofM. incognita adult females. The antibodies also reacted with secretory granules in both types of esophageal glands in M. javanica and M. arenaria J2 but not with granules in esophageal glands of Heterodera glycines J2.     

Suppression Mechanisms of Meloidogyne arenaria Race 1 by Pasteuria penetrans

The biological control of Meloidogyne arenaria on peanut (Arachis hypogaea) by Pasteuria penetrans was evaluated using a six x six factorial experiment in field microplots over 2 years. The main factors were six inoculum levels of second-stage juveniles (J2) of M. arenaria race 1 (0, 40, 200, 1,000, 5,000, and 25,000 J2/microplot, except that the highest level was 20,000 J2/microplot in 1995) and six infestation levels of P. penetrans as percentages of J2 with endospores attached (0, 20, 40, 60, 80, and 100%). The results were similar in 1994 and 1995. Numbers of eggs per root system, J2 per 100 cm³ soil at harvest, root galls, and pod galls increased with increasing nematode inoculum levels and decreased with increasing P. penetrans infestation levels (P ≤ 0.05), except that there was no effect of P. penetrans infestation levels on J2 per 100 cm³ soil in 1994 (P> 0.05). There were no statistical interaction effects between the inoculum levels of J2 and the infestation levels of P. penetrans (P > 0.05). When the infestation level was increased by 10%, the number of eggs per root system, root galls, and pod galls decreased 7.8% to 9.4%, 7.0% to 8.5%, and 8.0% to 8.7% in 1994 and 1995, respectively, whereas J2 per 100 cm³ soil decreased 8.8% in 1995 (P ≤ 0.05). The initial infestation level of P. penetrans contributed 81% to 95% of the total suppression of pod galls, whereas the infection of J2 of the subsequent generations contributed only 5% to 19% suppression of pod galls. The major suppressive mechanism of M. arenaria race 1 by P. penetrans on peanut is the initial endospore infestation of J2 at planting.     

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.     

Origin of a Meloidogyne incognita Surface Coat Antigen

The surface coat (SC) of plant nematodes is thought to originate either from the living hypodermis or from secretory glands associated with the excretory system or nervous system. In this study, we investigated the origin of the SC of Meloidogyne incognita by immunolocalization with a monoclonal antibody raised against the surface coat of the preparasitic juveniles (J2). Under the electron microscope, strong labeling was found on the cuticular surface and in the rectal dilation of the J2, while labeling was absent in other parts of the nematode, including the hypodermis, excretory system, nervous system, and digestive system. Because the rectal glands are known to be the origin of the gelatinous egg matrix produced by adult females of Meloidogyne, we also examined sections of mature females from monoxenic cultures of Arabidopsis thaliana. Labeling of the female occurred in the rectal glands and in the gelatinous matrix exuded from the anus. At the ultrastructural level, gold particles were mainly deposited in multivesicular bodies that appeared to be associated with the Golgi bodies of the rectal glands. Our results suggest that at least one component of the J2 SC originates from the rectal gland cells and that the SC of the J2 shares common epitopes with the gelatinous egg matrix of mature females.     

Influence of Initial Population Densities of Meloidogyne incognita on Three Chile Cultivars

The effects of Meloidogyne incognita on the Big Jim, Jalapeno, and New Mexico No. 6 chile (Capsicum annuum) cultivars were investigated in microplots for two growing seasons. All three cultivars were susceptible to M. incognita and reacted similarly to different initial populations of this nematode. Severe stunting and yield suppressions occurred at all initial M. incognita densities tested ranging from 385 to 4,230 eggs and larvae/500 cm³ soil. Regression analysis of the microplot data from a sandy loam soil showed yield losses of 31% for the 1978 season and 25% for the 1979 season for the three cultivars for each 10-fold increase in the initial population of M. incognita.     

Population Development of Pasteuria penetrans on Meloidogyne arenaria

A microplot study on the influence of cropping sequences with peanut in summer and bare fallowed or cover crops of rye or vetch in winter on the population development of Pasteuria penetrans was initiated in the spring of 1987. The number of spores of P. penetrans attached per second-stage juvenile of Meloidogyne arenaria race 1 increased from 0.11 in the fall of 1987 to 7.6, 8.6, and 3.6 in the fall of 1989 in the rye, vetch, and fallowed plots, respectively. Higher (P ≤ 0.05) levels of P. penetrans occurred in the rye and vetch plots than in fallowed plots. No influence of P. penetrans on peanut, rye, or vetch yield was observed in 1987 and 1988, but in 1989 peanut yield was 64% higher (P ≤ 0.05) in plots infested with P. penetrans than in plots without P. penetrans. Numbers of M. arenaria in plots without P. penetrans were influenced by the cropping sequences in the spring of 1988 and 1989 but not in the fall following the peanut crop. In the spring the plots with rye had the lowest nematode numbers in either year (P ≤ 0.05). Nematode numbers were lower (P ≤ 0.05) in plots with P. penetrans than in plots without P. penetrans in the spring of 1989 (vetch) and the fall of 1989 (rye, vetch, and fallowed).     

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.     

Quantification of Endospore Concentrations of Pasteuria penetrans in Tomato Root Material

Six methods for quantification of the endospore concentrations of Pasteuria penetrans from tomato roots are described. Mortar disruption and machine disruption methods gave the highest estimations (endospores per gram of root material) of 83.7 and 79.0 million, respectively. These methods were significantly superior to incubation bioassay (47.7 million), enzymatic disruption (32.1 million), and enzymatic disruption + flotation (25.8 million) methods. A centrifugation bioassay method gave the lowest estimation of 12.7 million.     

Survival of Paecilomyces lilacinus in Selected Carriers and Related Effects on Meloidogyne incognita on Tomato

Laboratory and microplot experiments were conducted to determine the influence of carrier and storage of Paecilomyces lilacinus on its survival and related protection of tomato against Meloidogyne incognita. Spores of P. lilacinus were prepared in five formulations: alginate pellets (pellets), diatomaceous earth granules (granules), wheat grain, soil, and soil plus chitin. Fungal viability was high in wheat and granules, intermediate in pellets, and low in soil and chitin-amended soil stored at 25 ± 2 C. In 1985 P. lilacinus in field microplots resulted in about a 25% increase in tomato yield and 25% gall suppression, compared with nematodes alone. Greatest suppression of egg development occurred in plots treated with P. lilacinus in pellets, wheat grain, and granules. In 1986 carryover protection of tomato against M. incognita resulted in about a threefold increase in tomato fruit yield and 25% suppression of gall development, compared with plants treated with nematodes alone. Higher numbers of fungus-infected egg masses occurred in plots treated with pellets (32%) than in those treated with chitin-amended soil (24%), wheat (16%), granules (12%), or soil (7%). Numbers of fungal colony-forming units per gram of soil in plots treated with pellets were 10-fold greater than initial levels estimated at planting time in 1986.     

Persistence and Suppressiveness of Pasteuria penetrans to Meloidogyne arenaria Race

The long-term persistence and suppressiveness of Pasteuria penetrans against Meloidogyne arenaria race 1 were investigated in a formerly root-knot nematode suppressive site following 9 years of continuous cultivation of three treatments and 4 years of continuous peanut. The three treatments were two M. arenaria race 1 nonhost crops, bahiagrass (Paspalum notatum cv. Pensacola var. Tifton 9), rhizomal peanut (Arachis glabrata cv. Florigraze), and weed fallow. Two root-knot nematode susceptible weeds commonly observed in weed fallow plots were hairy indigo (Indigofera hirsuta) and alyce clover (Alysicarpus vaginalis). The percentage of J2 with endospores attached reached the highest level of 87% in 2000 in weed fallow, and 63% and 53% in 2002 in bahiagrass and rhizomal peanut, respectively. The percentage of endospore-filled females extracted from peanut roots grown in weed fallow plots increased from nondetectable in 1999 to 56% in 2002, whereas the percentages in bahiagrass and rhizomal peanut plots were 41% and 16%, respectively. Over 4 years, however, there was no strong evidence that endospores densities reached suppressive levels because peanut roots, pods, and pegs were heavily galled, and yields were suppressed. This might be attributed to the discovery of M. javanica infecting peanut in this field in early autumn 2001. A laboratory test confirmed that although the P. penetrans isolate specific to M. arenaria attached to M. javanica J2, no development occurred. In summary, P. penetrans increased on M. arenaria over a 4-year period, but apparently because of infection of M. javanica on peanut at the field site root-knot disease was not suppressed. This was confirmed by a suppressive soil test that showed a higher level of soil suppressiveness than occurred in the field (P ≤ 0.01).     

Cuticular Collagenous Proteins of Second-stage Juveniles and Adult Females of Meloidogyne incognita: Isolation and Partial Characterization

Cuticles isolated from second-stage juveniles and adult females of Meloidogyne incognita were purified by treatment with 1% sodium dodecyl sulfate (SDS). The juvenile cuticle was composed of three zones differing in their solubility in β-mercaptoethanol (BME). Proteins in the cortical and median zones were partially soluble in BME, whereas the basal zone was the least soluble. The BME-soluble proteins from the juvenile cuticle were separated into 12 bands by SDS-polyacrylamide gel electrophoresis and characterized as collagenous proteins based on their sensitivity to collagenase and amino acid composition. The adult cuticle consisted of two zones which were dissolved extensively by BME. The basal zone was completely solubilized, leaving behind a network of fibers corresponding to the cortical zone. The BME-soluble proteins from the adult cuticle were separated by electrophoresis into nine bands one of which constituted > 55% of the total BME-soluble proteins. All bands were characterized as collagenous proteins. Collagenous proteins from juvenile cuticles also contained glycoproteins which were absent from the adult cuticles.