Detection and characterization of Pasteuria 16S rRNA gene sequences from nematodes and soils.

Various bacterial species in the genus Pasteuria have great potential as biocontrol agents against plant-parasitic nematodes, although study of this important genus is hampered by the current inability to cultivate Pasteuria species outside their host. To aid in the study of this genus, an extensive 16S rRNA gene sequence phylogeny was constructed and this information was used to develop cultivation-independent methods for detection of Pasteuria in soils and nematodes. Thirty new clones of Pasteuria 16S rRNA genes were obtained directly from nematodes and soil samples. These were sequenced and used to construct an extensive phylogeny of this genus. These sequences were divided into two deeply branching clades within the low-G + C, Gram-positive division; some sequences appear to represent novel species within the genus Pasteuria. In addition, a surprising degree of 16S rRNA gene sequence diversity was observed within what had previously been designated a single strain of Pasteuria penetrans (P-20). PCR primers specific to Pasteuria 16S rRNA for detection of Pasteuria in soils were also designed and evaluated. Detection limits for soil DNA were 100-10,000 Pasteuria endospores (g soil)(-1).

Environmental quantification of Pasteuria penetrans endospores using in situ antigen extraction and immunodetection with a monoclonal antibody.

Abstract Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.) that has attracted significant attention as a promising biocontrol agent. The inability to culture P. penetrans has invoked the need for a quantitative detection capability to facilitate biocontrol studies. A chemical extraction method using urea, dithiothreitol and CHES buffer (UDC) is shown to release soluble endospore envelope antigen from endospores present in complex matrices, generating an extract that can be used to determine the levels of spores when compared to a standard in an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody, MAb 2A41D10. Extractions can be performed in less than 1 h. Linear regression analysis routinely produced line fits with r(2)>0.90. Antigen extraction efficiency was not influenced by soil type. Three ELISA formats were analyzed for quantitative detection of P. penetrans endospores. A tertiary ELISA immunodetection system provided the lowest level of detection at approximately 300 spores per gram of soil. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots of soil extracts containing P. penetrans endospore antigen produced signature peptides bearing a common epitope characteristic of endospores of Pasteuria spp. MAb 2A41D10 was specific for Pasteuria spp. and did not react with extracts of Pasteuria-free soil or with spore extracts of native Gram-positive endospore-forming bacteria. Immunofluorescent microscopy revealed that MAb 2A41D10 recognizes an epitope uniformly distributed on the endospore surface. The development of a rapid extraction method and analysis of solubilized antigen by immunodetection has the potential for broad application in food and environmental microbiology.

Ultrastructure and Development of Pasteuria sp. (S-1 strain), an Obligate Endoparasite of Belonolaimus longicaudatus (Nemata: Tylenchida).

Pasteuria sp., strain S-1, is a gram-positive, obligate endoparasitic bacterium that uses the phytoparasitic sting nematode, Belonolaimus longicaudatus, as its host in Florida. The host attachment of S-1 appears to be specific to the genus Belonolaimus with development occurring only in juveniles and adults of B. longicaudatus. This bacterium is characterized from other described species of Pasteuria using ultrastructure of the mature endospore. Penetration, development, and sporogenesis were elucidated with TEM, LTSEM, and SEM and are similar to other nematode-specific Pasteuria. Recent analysis of 16S rDNA sequence homology confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans, and corroborates ultrastructural, morphological, morphometric, and host-range evidence suggesting separate species status.

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.

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 cm(3) 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). 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 cm(3) soil decreased 8.8% in 1995 (P

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.

Response of Meloidogyne spp., Heterodera glycines, and Radopholus similis to Tannic Acid.

Tannins, which are water-soluble polyphenols, are toxic to numerous fungi, bacteria, and yeasts. Our objectives were to study the efficacy of tannic acid in control of Meloidogyne arenaria on tomato and its effects on the behavior of M. arenaria, M. incognita, Heterodera glycines, and Radopholus similis. Three concentrations of tannic acid, 0.1, 1.0, and 10 g/500 cm(3) of soil, were applied preplant (powder) and at-plant (powder and drench) into soil infested with M. arenaria. Tannic acid at the 1.0-g rate reduced galling compared with the untreated control, regardless of methods of application. The 0.1-g rate resulted in no reduction in galling when applied preplant but reduced galling when applied as a drench and in one of two experiments when applied at-plant. The 10-g rate was phytotoxic to tomato seedlings except when applied 7 days preplant. In the latter case, root galling was suppressed to very low numbers. In behavior studies on water agar, Meloidogyne second-stage juveniles were attracted to areas with an increasing tannic acid gradient. Radopholus similis was repelled from the tannic acid gradient in one of two experiments. There was no effect on H. glycines. The response of M. arenaria second-stage juveniles to different concentrations of tannic acid dissolved in alginate was tested. Movement behavior of the second-stage juveniles were observed at 1,000 and 10,000 mug/ml of tannic acid, but not at 10 and 100 mug/ml.

Tannic Acid Effects on Hatching of Heterodera glycines in Vitro.

Effects of tannic acid on hatching of Heterodera glycines eggs were determined in vitro using three batches of eggs obtained from greenhouse cultures in Florida or from naturally infested field soil in Minnesota. A quadratic model fits the percentage egg hatch. Hatch increased with increasing tannic acid concentrations from 0 to about 39 mg/liter, then declined with further increases in concentration. Tannic acid did not induce hatching of dormant eggs obtained from the field.

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.

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.

Effects of Tropical Rotation Crops on Meloidogyne arenaria Population Densities and Vegetable Yields in Microplots.

The effects of 12 summer crop rotation treatments on population densities of Meloidogyne arenaria race 1 and on yields of subsequent spring vegetable crops were determined in microplots. The crop sequence was: (i) rotation crops during summer 1991 ; (ii) cover crop of rye (Secale cereale) during winter 1991-92; (iii) squash (Cucurbita pepo) during spring 1992; (iv) rotation crops during summer 1992; (v) rye during winter 1992-93; (vi) eggplant (Solanum melongena) during spring 1993. The 12 rotation treatments were castor (Ricinus communis), cotton (Gossypium hirsutum), velvetbean (Mucuna deeringiana), crotalaria (Crotalaria spectabilis), fallow, hairy indigo (Indigofera hirsuta), American jointvetch (Aeschynomene americana), sorghum-sudangrass (Sorghum bicolor x S. sudanense), soybean (Glycine max), horsebean (Canavalia ensiformis), sesame (Sesamum indicum), and peanut (Arachis hypogaea). Compared to peanut, the first eight rotation treatments resulted in lower (P

Occurrence of Pasteuria spp. in Florida.

Two years of data collected from the Florida Nematode Assay Laboratory of the Florida Cooperative Extension Service and 4 years of data from the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, were compiled to find out the distribution of Pasteuria spp. on nematodes in Florida soils. Information recorded came from 335 samples and included nematode genera with Pasteuria endospores attached, host plants associated with the samples, and the origins of the samples. Pasteuria spp. were detected on 14 different plant-parasitic nematode genera in 41 Florida counties and associated with over 39 different plant species and in seven fallow fields. Pasteuria-infected nematodes were associated with a wide range of plant hosts, although frequency of associations with these hosts reflected the sample bias of the laboratories involved. Meloidogyne and Hoplolaimus spp. were the two nematode genera most frequently associated with Pasteuria. Pasteuria spp. were observed attached to members of these two genera in 176 and 59 soil samples, respectively.

A Centrifugation Method for Attaching Endospores of Pasteuria spp. to Nematodes.

Attachment of relatively low numbers of endospores from two isolates of Pasteuria spp. to several species of nematodes was consistently achieved in 2-5 minutes with a centrifugation technique. The rate of attachment of Pasteuria penetrans at 10 endospores/0.1 ml/tube to second-stage juveniles (J2) of Meloidogyne javanica, M. incognita race 1, M. incognita race 3, and M. arenaria races 1 and 2 in two tests averaged 4.4, 5.2, 0.1, 0.3, and 0 endospores per J2, respectively. The rate of attachment Pasteuria sp. at 10(3) endospores/0.1 ml/tube to individuals of Hoplolaimus galeatus, Belonolaimus longicaudatus, M. arenaria race 1, M. javanica, and M. incognita race 1 in two tests averaged 0.8, 0.04, 0, 0, and 0 endospores per nematode, respectively. The rate of attachment of P. penetrans to M. javanica at 10(3), 10, or 10 endospores/0.1 ml/tube from two tests averaged 1.0, 5.7, and 28.3 endospores per J2, respectively. All of the J2 had endospores attached following centrifugation in tubes with 10 and 10 endospores/0.1 ml/tube.

Damage Functions for Meloidogyne arenaria on Peanut.

Microplot experiments were conducted in 1989 and 1990 to determine the relationship between yield of peanut (Arachis hypogaea) and inoculum density ofMeloidogyne arenaria race 1. Nine inoculum densities were used, ranging from 0-200 eggs/100 cm(3) soil (1989) or from 0-100 eggs/100 cm(3) (1990), and each density was replicated 10 times. In 1989, higher final densities (mean of 1,171 juveniles [J2]/100 cm(3) soil) were obtained in plots inoculated with 0.5 to 50 eggs/100 cm(3) soil than in plots inoculated with 100 to 200 eggs/100 cm(3) (313 J2/100 cm(3) soil). In 1990, final densities of M. arenaria reached high levels (>/= 1,111 J2/100 cm(3) soil) in all inoculated plots. Pod yield and dry weight of foliage at harvest were negatively correlated (P

Comparisons of Isozyme Phenotypes in Five Meloidogyne spp. with Isoelectric Focusing.

Meloidogyne incognita race 1, M. javanica, M. arenaria race 1, M. hapla, and an undescribed Meloidogyne sp. were analyzed by comparing isozyme phenotypes of esterase, malate dehydrogenase, phosphoglucomutase, isocitrate dehydrogenase, and alpha-glycerophosphate dehydrogenase. Isozyme phenotypes were obtained from single mature females by isoelectric focusing electrophoresis. Of these five isozymes, only esterase and phosphoglucomutase could be used to separate all five Meloidogyne spp.; however, the single esterase electromorphs were similar for M. incognita and M. hapla. Yet when both nematodes were run on the same gel, differences in their esterase phenotypes were detectable. Isozyme phenotypes from the other three isozymes revealed a great deal of similarity among M. incognita, M. javanica, M. arenaria, and the undescribed Meloidogyne sp.

Specific Gravity of Spores of Pasteuria penetrans and Extraction of Spore-filled Nematodes from Soil.

The specific gravity of spores of Pasteuria penetrans collected from Meloidogyne arenaria was found to be around 1.28. Increasing the sucrose concentration used for the extraction of Pratylenchus scribneri from a specific gravity of 1.14 to 1.26 led to the recovery of higher numbers of specimens filled with spores of Pasteuria sp. (P

Effects of Bahiagrass and Nematicides on Meloidogyne arenaria on Peanut.

A field infested with Meloidogyne arenaria and with a history of peanut yield losses was divided into two equal parts. One-half of the field (bahia site) was planted to bahiagrass in 1986 and maintained through 1987. The other half (peanut site) was planted to soybean in 1986 and peanut in 1987 with hairy vetch planted each fall as a cover crop. In 1988 identical nematicide treatments including 1,3-dichloropropene (1,3-D), aldicarb, and ethoprop were applied to the two sites, and the sites were planted with the peanut cultivar Florunner. At mid-season, population levels of M. arenaria second-stage juveniles in the bahia site were relatively low, compared with those in the peanut site. At harvest, however, population levels were high in both sites. No nematicide treatment increased yields over the untreated control in either site (P

Evaluation of Paecilomyces lilacinus as a Biocontrol Agent of Meloidogyne javanica on Tobacco.

The efficacy of the nematode parasite Paecilomyces lilacinus, alone and in combination with phenamiphos and ethoprop, for controlling the root-knot nematode Meloidogyne javanica on tobacco and the ability of this fungus to colonize in soil under field conditions were evaluated for 2 years in microplots. Combinations and individual treatments of the fungus grown on autoclaved wheat seed, M. javanica eggs (76,000 per plot), and nematicides were applied to specified microplots at the time of transplanting tobacco the first year. Vetch was planted as a winter cover crop, and the fungus and nematicides were applied again the second year to specified plots at transplanting time. The fungus did not control the nematode in either year of these experiments. The average root-gall index (0 = no visible galls and 5 = > 100 galls per root system) ranged from 2.7 to 3.9 the first year and from 4.3 to 5.0 the second in nematode-infested plots treated with nematicides. Plants with M. javanica alone or in combination with P. lilacinus had galling indices of 5.0 both years; the latter produced lower yields than all other treatments during both years of the study. Nevertheless, the average soil population densities of P. lilacinus remained high, ranging from 1.2 to 1.3 x 106 propagules/g soil 1 week after the initial inoculation and from 1.6 to 2.3 x 104 propagules/g soil at harvest the second year. At harvest the second year the density of fungal propagules was greatest at the depth of inoculation, 15 cm, and rapidly decreased below this level.

Efficacy of Fumigant and Nonfurmigant Nematicides for Control of Meloidogyne arenaria on Peanut.

Three tests were conducted to evaluate the efficacy of fumigant and nonfumigant nematicides for control of Meloidogyne arenaria race 1 on peanut. Methyl bromide, 1,3-D, methyl isothiocyanate, and methyl isothiocyanate mixtures were applied 7 or 8 days preplant either broadcast or in-the-row. Aldicarb, ethoprop, fenamiphos, and F5145 were applied at different rates and by different methods at-plant or at early flowering. Of the 32 treatments evaluated, only seven resulted in yield increases (P = 0.05), although early season vigor was high in all treated plots. During the latter one-third of the growing season, however, nematode control was not adequate in most treatments resulting in heavy peg, pod, and root infection by M. arenaria.

Tillage and multiple cropping systems and population dynamics of phytoparasitic nematodes.

The effect of two cropping and tillage systems on the population dynamics of four nematode species was evaluated on a loamy sand. Hairy vetch succeeded by corn or grain sorghum was seeded in split plots randomized within whole plots of no-tillage versus conventional tillage over four growing seasons (1980-83). The vetch-corn cropping system increased the density of Meloidogyne incognita 2.9 x more than the vetch-grain sorghum cropping system. In contrast, the vetch-grain sorghum cropping system increased the density of Criconemella ornata 0.7 x more than the vetch-corn cropping system. Meloidogyne incognita and C. ornata were affected more by these cropping systems than were Pratylenchus brachyurus or Paratrichodorus minor. Multiple cropping systems, vetch varieties, and crop host preference affected nematode population densities, whereas tillage treatments, conventional or no-tillage, had little effect on them.