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

Abstract

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 is a gram positive hyperparasite of root-knot nematodes (RKN: Meloidogyne spp.) and other nematodes. This bacterium is a member of the Bacillus-Clostridium clade, and it is most closely related to Bacillus halodurans and B. subtilis (Charles et al., 2005). In nature, endospores of P. penetrans attach to the cuticle of second-stage juveniles (J2) migrating through soil. Pasteuria penetrans endospores germinate when the nematode establishes a feeding site in the host vasculature (Sayre, 1993). A germ tube is extended into the pseudocoelom of the nematode, which gives rise to microcolonies, thalli, and, eventually, endospores. Proliferation of the endoparasite within the pseudocoelomic cavity of the nematode causes degeneration of the reproductive tissues, greatly reducing fecundity. Endospores are released into the surrounding soil upon decay of the female cadavers and root tissue. Due to its ability to control nematode populations, P. penetrans has potential as a biocontrol agent for RKN (Chen and Dickson, 1998). Although the use of P. penetrans to control RKN is promising, its fastidious nature has inhibited mass production of endospores.

Current methods for collecting endospores of P. penetrans require handpicking infected females (Oostendorp et al., 1990; Chen et al., 2000). Here we present a rapid method for collecting endospores that eliminates the need for handpicking females. Our method includes mechanical processing of digested root material and filtration to collect quantities of viable P. penetrans endospores orders of magnitude higher than previously reported methods.

Materials and Methods

Pasteuria penetrans (Gainesville, Florida, Isolate; Pasteuria Bioscience, Alachua, FL) endospores were attached to Meloidogyne arenaria Race 1 J2 using a modified method of Hewlett and Dickson (1993). Briefly, endospores of P. penetrans were attached to 10,000 J2 at a ratio of 100 endospores/J2 by centrifugation at 3,220g for 5 min at room temperature in 10 ml distilled water in a 50-ml tube. Average attachment, average number of endospores attached to nematodes, and percentage attachment were determined. Ten thousand J2, with 97% ± 6% (mean ± 1 standard deviation for three replicates) having endospores attached, were used to inoculate each 4-wk-old tomato plant (Lycopersicon esculentum ‘Rutgers large red’) maintained at approximately 30°C in a greenhouse. After about 60 d, roots were harvested from the plant, rinsed with room temperature water, and soaked for 4 hr at room temperature or overnight at 4°C in a 1:5 dilution of 100X Crystalzyme (Valley Research, South Bend, IN) pectinase solution.

Softened galls (with excess root material removed) were placed in a mortar and gently smashed with a pestle to crush galls and, consequently, endospore-filled females. The smashed root material was transferred to a glass jar filled with 50 ml distilled water and then shaken for 2 min. Endospores were collected by pouring the resultant slurry over a series of stacked sieves: a 250-μm-pore sieve, a 75-μm-pore sieve, and a 25-μm-pore sieve on the bottom. The material passing through the 25-μm-pore sieve was recovered and transferred to a glass jar, and the shaking step was repeated once with an additional 50 ml distilled water. The slurry was again filtered through the series of stacked sieves and the eluate filtered through a 5-μm polycarbonate filter (GE Osmonics, Minnetonka, MN) under vacuum; the filtrate was recovered and stored at 4°C. This sample was visually inspected for the presence of endospores using a light microscope at ×400. The endospore concentration was determined using a Brightline hemacytometer (Hausser Scientific, Horsham, PA). Differential interference images were collected using a Leica DM1RB microscope with a ×100 oil immersion objective (1.3 NA).

The viability of endospores collected using the present (mortar-ground-gall) method was assessed by attachment to and infection of M. arenaria Race 1 J2. For infectivity assays, 10,000 J2 with endospores attached (from the present method or handpicked females disrupted in a glass homogenizer) were used to inoculate tomato plants. After approximately 60 d, 20 females were arbitrarily handpicked from digested root material, visually inspected for endospores, and crushed in distilled water with a glass tissue grinder to release endospores.

Endospore DNA was extracted after pelleting 5 × 10⁷ endospores at 18,000g for 5 min. The endospores were suspended in 10 mM Tris, 1.0 mM EDTA, pH 8.0, containing 20 mg/ml lysozyme, and incubated at 37°C for 30 min. Proteinase K (20 mg/ml) and sodium dodecyl sulfate (2% w/v) were added followed by additional 30 min incubation. The pretreated endospores were transferred to a 2.0-ml impact-resistant tube containing approximately 1 g glass beads in the size range 150 to 212 μm (Sigma-Aldrich, St. Louis, MO). Phenol and chloroform were added, and the endospores were disrupted using a Savant FastPrep Bio101 bead beating instrument. The samples were extracted with four 30-sec pulses at 4.0 m/s interspersed with 1 min incubations on ice. The phases were separated by centrifugation at 18,000g for 10 min. The aqueous phase was further purified with subsequent phenol and chloroform extractions. Endospore DNA was precipitated by adding sodium acetate, pH 4.8, to 300 mM, 2.5 volumes 95% ethanol, and 0.014 mg/ml glycogen, followed by centrifugation at 20,000g for 60 min at 4°C. RNase One (Promega, Madison, WI) was used to remove contaminating RNA per manufacturer recommendations. Endospore DNA concentration was determined fluorometrically. To confirm that usable genomic DNA could be isolated from spores extracted with this method, PCR was performed on DNA extracted from endospores using Pasteuria 16S ribosomal DNA primers (Duan et al., 2003). Experiments were repeated twice for a total of three trials.

Results and Discussion

Davies et al. (1988) found that attachment of at least 5 endospores/J2 was necessary to ensure infection of the nematode by P. penetrans, but that subsequent plant-root penetration was reduced by 86% when J2 were encumbered with 15 or more endospores. In this study, we inoculated plants with 10,000 J2 with an average of six endospores attached. Pasteuria-infected females produce approximately 2 × 10⁶ endospores each. Therefore, a plant infected with 10,000 Pasteuria-filled females contains up to 2 × 10¹⁰ endospores. Using our method, we were able to collect an average of 5.78 × 10⁹ endospores per plant, which is approximately 30% of that theoretical maximum. This is a significant improvement over an older mortar disruption method for isolating endospores (Chen et al., 1996), which did not include enzymatic digestion or filtration to remove plant and/or nematode debris and produced 1.67 × 10⁸ endospores/plant, or about 30-fold fewer than our method.

By eliminating the need for handpicking endospore-filled females, our method greatly reduced endospore collection time and increased the number of viable endospores that may be collected. The present method has been optimized for collecting endospores from the roots of three plants in 1 hr. However, it can easily be used to collect endospores from several more plants with a negligible increase in time.

Although noninfected and Pasteuria-filled M. arenaria females were not initially separated, passing the endospores through a 5-μm filter effectively removed most nematode and plant debris. Visual inspection of endospores collected using the present method revealed endospores with normal morphology and only a small amount of nematode and/or root debris (Fig. 1). Exosporium, spore core, and parasporal fibers are present and intact. For more sensitive assays, such as antiserum production, subsequent purification of endospores may be necessary.

Fig. 1.

Nomarski differential interference contrast microscopy of the Pasteuria penetrans endospore filtrate. Scale bar = 10 μm.

Endospores collected by our method retained full biological activity and effectively attached to and infected M. arenaria J2. There were no differences in the average attachment or percentage attachment of endospores collected using the present method when compared to endospores from handpicked females. Average attachment was 5.6 ± 3.4 and 6.0 ± 3.1 endospores (mean ± 1 standard deviation for three replicates) per J2 for crushed galls or handpicked females, respectively. Likewise, there were no detectable differences in the infectivity of endospores collected using the present method vs. those from handpicked females. There was 95% and 97% average infectivity of J2 with endospores prepared with the present method and from 20 handpicked crushed females, respectively.

Polymerase chain reaction analysis with primers specific for Pasteuria 16S ribosomal DNA sequences yielded the anticipated 549-bp band. Genes isolated in this way are suitable for cloning and sequencing (Anderson et al., 1999; Atibalentja et al., 2000; Trotter and Bishop, 2003). The present method is ideal for rapidly collecting vast amounts of viable endospores suitable for attaching to and infecting nematodes, morphology studies, and DNA preparations.