Potential for Misidentification of a Spore-Forming Paenibacillus polymyxa Isolate as an Endophyte by Using Culture-Based Methods

While Paenibacillus polymyxa strain Pw-2 has been identified as an endophyte of lodgepole pine (M. Shishido, B. M. Loeb, and C. P. Chanway, Can. J. Microbiol. 41:707-713, 1995), P. polymyxa strain L6 has not, a distinction that could be explained by the differential abilities of these isolates to form spores, rather than the differential abilities to colonize the interior tissues of lodgepole pine. Chemical disinfection was used to destroy bacteria on the root exterior, but bacterial endospores are known for their ability to withstand chemical disinfection, and strain Pw-2 was found to produce 300 to 11,000 times more germinating endospores than strain L6 under the experimental conditions used by Shishido et al. (Can. J. Microbiol. 41:707-713, 1995). Attempts to identify strain Pw-2 within lodgepole pine root tissues by using confocal microscopy techniques failed. We discuss the possibility that spore-forming bacteria can be mistakenly identified as endophytes when culture-based methods alone are used.     

Alterations in plant growth and in root hormone levels of lodgepole pines inoculated with rhizobacteria.

The presence of other soil microorganisms might influence the ability of rhizobacterial inoculants to promote plant growth either by reducing contact between the inoculant and the plant root or by interfering with the mechanism(s) involved in rhizobacterially mediated growth promotion. We conducted the following experiments to determine whether reductions in the extent of growth promotion of lodgepole pine mediated by Paenibacillus polymyxa occur in the presence of a forest soil isolate (Pseudomonas fluorescens M20) and whether changes in plant growth promotion mediated by P. polymyxa (i) are related to changes in P. polymyxa density in the rhizosphere or (ii) result from alterations in root hormone levels. The extent of plant growth, P. polymyxa rhizosphere density, and root hormone concentrations were determined for lodgepole pine treated with (i) a single growth-promoting rhizobacterial strain (P. polymyxa L6 or Pw-2) or (ii) a combination of bacteria: strain L6 + strain M20 or strain Pw-2 + strain M20. There was no difference in the growth of pines inoculated with strain L6 and those inoculated with strain L6 + strain M20. However, seedlings inoculated with strain Pw-2 had more lateral roots and greater root mass at 12 weeks after inoculation than plants inoculated with strain Pw-2 + strain M20. The extent of growth promotion mediated by P. polymyxa L6 and Pw-2 in each treatment was not correlated to the average population density of each strain in the rhizosphere. Bacterial species-specific effects were observed in root hormone levels: indole-3-acetic acid concentration was elevated in roots inoculated with P. polymyxa L6 or Pw-2, while dihydrozeatin riboside concentration was elevated in roots inoculated with P. fluorescens M20.     

Surface colonization of lodgepole pine (Pinus contorta var. latifolia [Dougl. Engelm.]) roots by Pseudomonas fluorescens and Paenibacillus polymyxa under gnotobiotic conditions

Indirect immunofluorescence techniques and confocal scanning laser microscopy were used to identify rhizobacterial strains on the root surfaces of pine seedlings, which were grown from seeds under gnotobiotic conditions. Conifer plant growth promoting rhizobacterial strains Paenibacillus polymyxa L6 and Pw-2, and the forest soil isolate Pseudomonas fluorescens M20, were inoculated onto surface-disinfested pine seeds, singly, or in dual combinations: strains L6 + M20, or strains Pw-2 + M20. Segments containing particular root microsites (root tip, root hair zone, or areas of lateral root emergence) were sampled randomly from roots 7 or 13 weeks after inoculation, and the colonization of roots by each bacterium was observed. Root segments were also sampled from individual roots at six different points along the length of the root, and the qualitative colonization of younger areas, closer to the root tip, contrasted with that of older areas, closer to the root base. The ability of strain M20 to colonize root areas adjacent to sites of lateral root emergence improves in the presence of either P. polymyxa strain, while the ability of the P. polymyxa strains to colonize these areas was not affected. More rhizobacteria were also generally observed on younger root tissues than on areas closer to the root base.     

Effect of Plant Growth Promoting Bacillus Strains on Pine and Spruce Seedling Growth and Mycorrhizal Infection

Rifamycin-resistant derivatives of plant growth promotingBacilluspolymyxa strains L6, Pw-2, and S20 were used to evaluate theinteraction of bacterial–mycorrhizal co-inoculation onpine and spruce seedling growth. We were particularly interestedin determining if the mechanism by which bacteria stimulatedseedling growth depended on the presence of ectomycorrhizae.Mycorrhizal inoculum was introduced by adding 2ml of one ofsix forest floor soil types originating from different spruceand pine stands to seedling containers. Mycorrhizal roots developedin 34% of pine and 27% of spruce seedlings treated with forestsoil, but no differences between forest soils were detected.Most mycorrhizae were formed byWilcoxinasp. (E-strain) (98%for spruce and 67% for pine); small numbers ofAmphinema-like,Myceliumradicis atrovirens, Suillus-like,Thelephora-like, andTuber-likemycorrhizae were also found on pine (27% in total).Thelephora-likefungi comprised 2% of spruce mycorrhize. In the absence of bacterialinoculum, spruce seedling biomass was positively correlatedwith the number of mycorrhizal root tips, but this trend wasnot detected in spruce inoculated with bacteria or in pine.Bacterial inoculation did not influence the mycorrhizal statusof seedlings, but all threeBacillusstrains stimulated growthof both conifer species. Root biomass, in particular, was significantlyenhanced by up to 18% compared with uninoculated controls. Mycorrhizalfungi improved the growth of spruce seedlings, but plant growthpromotion byBacilluswas similar for mycorrizal and non-mycorrhizalseedlings of both species. Our results suggest thatBacillusstrainsL6-16R, Pw-2R, and S20-R enhance conifer seedling growth througha mechanism unrelated to mycorrhizal fungi. Hybrid spruce; Picea glaucaxengelmannii ; lodgepole pine; Pinus contortavar.latifoliaEngelm.; inoculation; Bacillus polymyxa; seedling growth promotion; mycorrhizae     

Cytometric monitoring of growth, sporogenesis and spore cell sorting in Paenibacillus polymyxa (formerly Bacillus polymyxa)

AIMS: Formation of bacterial endospores is a basic process in Gram-positive bacteria and has implications for health, industry and the environment. Flow cytometry offers a practical alternative for the rapid detection, enumeration and characterization of bacterial endospores. METHODS AND RESULTS: Paenibacillus polymyxa was chosen for this study because its spores cause sporangium deformation and have thick walls with a star-shaped section. Sporulating populations were analysed with a particle analyser and a flow cytometer after labelling with propidium iodide and Syto-13. Flow cytometric detection of single spores was confirmed by optical and scanning electron microscopy after cell sorting. Four cell sub-populations were cytometrically detected in P. polymyxa cultures grown in liquid sporulation medium. Two sub-populations consisted of vegetative cells differing in both morphology and viability; the other two sub-populations consisted of spores differing in their viability. CONCLUSIONS: This work has shown that flow cytometry is a simple and fast method (less than 15 minutes for sample preparation and analysis) for the study of the sporulation in P. polymyxa. The use of this technique allowed both detection and quantification of sporulation inside a culture, and distinguished cells that differed in viability despite being morphologically identical under microscopic observation. SIGNIFICANCE AND IMPACT OF THE STUDY: Flow cytometry has been proved to be a valuable tool for the analysis of sporulation in P. polymyxa cultures, with the unique capacity of distinguishing between endospores and vegetative cells, and between live and dead cells, in the same analysis. An important percentage of non-viable endospores has been found in aged cultures using this method.     

Asynchrony in larval development of the pine beauty moth, Panolis flammea, on an introduced host plant may affect parasitoid efficacy

In the United Kingdom, Panolis flammea (Den. and Schiff.) (Lepidoptera: Noctuidae) is an important pest species of the introduced lodgepole pine but not of its natural host Scots pine. The timing of P. flammea larval growth must be synchronized with its host tree if the larvae are to succeed. We collected field data during 1990 which revealed that the phenological window starts earlier in Scots pine and is shorter than that observed in lodgepole pine. The larvae are found in the field earlier and within a narrower time frame within a Scots pine forest than in a lodgepole pine forest. The larval developmental period is significantly longer on lodgepole pine than on Scots pine. The synchrony/asynchrony of P. flammea to its natural host (Scots pine) and an introduced tree (lodgepole pine) results in the parasitoids having a different impact on the larvae of the two hosts. At any one time, the host plant, caterpillars and parasitoids are more synchronous on the ancestral Scots pine than on lodgepole pine, resulting in a higher percentage of larvae in the optimal instar for parasitism at that time. In lodgepole pine, the percentage of suitable instars available to parasitoids is lower at any given time. The information presented here furthers our understanding of the possible mechanisms for the observed differential population dynamics of the insect on Scots pine and lodgepole pine in the UK. Handling editor: Robert Glinwood.     

Comparison of enrichment broths for isolation of Campylobacter jejuni.

Growth of Campylobacter jejuni was compared in enrichment broths of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) and Park et al. (C. E. Park, Z. K. Stankiewicz, J. Lovett, and J. Hunt, Can. J. Microbiol. 27:841-842, 1981), as modified by Lovett et al. (J. Lovett, D. W. Francis, and J. M. Hunt, Appl. Environ. Microbiol. 46:459-462, 1983). Inoculated foods used were cream-pudding types, which may be cross-contaminated by improper handling, improper storage of meats prepared simultaneously, or the use of raw milk as an ingredient. Both broths adequately supported growth.     

Comparison of enrichment broths for isolation of Campylobacter jejuni

Growth of Campylobacter jejuni was compared in enrichment broths of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) and Park et al. (C. E. Park, Z. K. Stankiewicz, J. Lovett, and J. Hunt, Can. J. Microbiol. 27:841-842, 1981), as modified by Lovett et al. (J. Lovett, D. W. Francis, and J. M. Hunt, Appl. Environ. Microbiol. 46:459-462, 1983). Inoculated foods used were cream-pudding types, which may be cross-contaminated by improper handling, improper storage of meats prepared simultaneously, or the use of raw milk as an ingredient. Both broths adequately supported growth.     

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.     

Changing dynamics of the pine beauty moth (Panolis flammea) in Britain: the loss of enemy free space?

1 The pine beauty moth Panolis flammea has two main host plants in Britain: Pinus sylvestris (Scots pine), which is the ancestral food plant where the insect is never abundant enough to cause tree mortality, and Pinus contorta (lodgepole pine), an introduced host tree that has experienced periodic widespread tree mortality due to this pest.
2 We review the recent literature, published mostly after the year 2000, regarding the impact of natural enemies on the population dynamics of P. flammea in Britain.
3 The natural enemies of P. flammea are more diverse and abundant in Scots pine habitat than in lodgepole pine habitat and some of them show differential selection for P. flammea larvae in Scots pine habitat over those located in lodgepole pine habitat.
4 It is concluded that the difference in the population dynamics of this insect in the two different habitats was probably the result of the P. flammea finding enemy-free space in lodgepole pine habitat.
5 Recent evidence on the diversity and impact of natural enemies on lodgepole pine has demonstrated that they currently have a much more significant impact on this pest than they did in the 1970s and 1980s, when outbreaks were frequent.     

The impact of phloem nutrients on overwintering mountain pine beetles and their fungal symbionts

In the low nutrient environment of conifer bark, subcortical beetles often carry symbiotic fungi that concentrate nutrients in host tissues. Although bark beetles are known to benefit from these symbioses, whether this is because they survive better in nutrient-rich phloem is unknown. After manipulating phloem nutrition by fertilizing lodgepole pine trees (Pinus contorta Douglas var. latifolia), we found bolts from fertilized trees to contain more living individuals, and especially more pupae and teneral adults than bolts from unfertilized trees at our southern site. At our northern site, we found that a larger proportion of mountain pine beetle (Dendroctonus ponderosae Hopkins) larvae built pupal chambers in bolts from fertilized trees than in bolts from unfertilized trees. The symbiotic fungi of the mountain pine beetle also responded to fertilization. Two mutualistic fungi of bark beetles, Grosmannia clavigera (Rob.-Jeffr. & R. W. Davidson) Zipfel, Z. W. de Beer, & M. J. Wingf. and Leptographium longiclavatum Lee, S., J. J. Kim, & C. Breuil, doubled the nitrogen concentrations near the point of infection in the phloem of fertilized trees. These fungi were less capable of concentrating nitrogen in unfertilized trees. Thus, the fungal symbionts of mountain pine beetle enhance phloem nutrition and likely mediate the beneficial effects of fertilization on the survival and development of mountain pine beetle larvae.     

Antibiosis plays a role in the context of direct interaction during antagonism of Paenibacillus polymyxa towards Fusarium oxysporum

Interaction of Fusarium oxysporum and Paenibacillus polymyxa starts with polar attachment of bacteria to the fungal hyphae followed by the formation of a large cluster of non-motile cells embedded in an extracellular matrix in which the bacteria develop endospores. Enumeration of fungal viable counts showed that less than one of 36,000 colony-forming units survived in paired cultures for 71 h. Effective antagonism was not observed below pH5 and was specific for the bacterial species. Development of F. oxysporum was inhibited in cell-free filtrates derived from cultures of P. polymyxa, but was much more strongly repressed in the presence of living bacteria. Furthermore, recovery of fungal growth started immediately after addition of antibiotics to paired cultures. Restoration of fungal growth was enhanced in filtrates that were supplemented with MgCl2, which suggests that anti-fungal compounds produced by the bacteria were counteracted by magnesium ions. In paired cultures, fungal counts remained very low, even in the presence of the magnesium salt. This study clearly showed that P. polymyxa antagonizes the plant pathogenic fungus F. oxysporum in liquid medium by means of an interaction process in which the presence of living bacteria is a prerequisite for continuous suppression of fungal growth.     

Sensitivity of soil bacteria isolated from the alienated zone around the Chernobyl Nuclear Power Plant to various stress factors

Seventy strains of chemoorganotrophic bacteria isolated by our group in 1993-1994 from soil sampled in the zone around the Chernobyl Nuclear Power Plant (ChNPP) were studied with respect to their sensitivity to various stress factors damaging DNA. Bacillus subtilis, B. cereus (both spores and vegetative cells), Methylobacterium extorquens, M. mesophilicum, and unidentified pigmented bacteria were found to be the most resistant to ultraviolet (UV) radiation, exhibiting LD90 values of 40 to more than 211 J/m2. The same bacteria, as well as Bacillus polymyxa, were tolerant to hydrogen peroxide (lethal concentrations of H2O2 ranged from 0.3 to 1.0 M); i.e., UV-resistant strains were also tolerant to hydrogen peroxide and vice versa. Fluorescent pseudomonads were the most sensitive to both UV radiation and H2O2, showing LD90 from 6 to 18 J/m2 and a lethal concentration of H2O2 lower than 0.1 M. All of the soil samples collected in the alienated zone around the ChNPP, where the radioactivity of the soil had decreased from 1000 to 2 microCi/kg soil over the period from 1987 to 1995, contained not only resistant bacteria but also a small number of bacteria sensitive to UV radiation and H2O2.     

A comparison of ammonium, nitrate and proton net fluxes along seedling roots of Douglas-fir and lodgepole pine grown and measured with different inorganic nitrogen sources

Significant spatial variability in NH4+, NO3- and H+ net fluxes was measured in roots of young seedlings of Douglas-fir (Pseudotsuga menziesii) and lodgepole pine (Pinus contorta) with ion-selective microelectrodes. Seedlings were grown with NH4+, NO3-, NH4NO3 or no nitrogen (N), and were measured in solutions containing one or both N ions, or no N in a full factorial design. Net NO3- and NH4+ uptake and H+ efflux were greater in Douglas-fir than lodgepole pine and in roots not exposed to N in pretreatment. In general, the rates of net NH4+ uptake were the same in the presence or absence of NO3-, and vice versa. The highest NO3- influx occurred 0-30 mm from the root apex in Douglas-fir and 0-10 mm from the apex in lodgepole pine. Net NH4+ flux was zero or negative (efflux) at Douglas-fir root tips, and the highest NH4+ influx occurred 5-20 mm from the root tip. Lodgepole pine had some NH4+ influx at the root tips, and the maximum net uptake 5 mm from the root tip. Net H+ efflux was greatest in the first 10 mm of roots of both species. This study demonstrates that nutrient uptake by conifer roots can vary significantly across different regions of the root, and indicates that ion flux profiles along the roots may be influenced by rates of root growth and maturation.     

Acid-catalyzed steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol

Utilization of ethanol produced from biomass has the potential to offset the use of gasoline and reduce CO(2) emissions. This could reduce the effects of global warming, one of which is the current outbreak of epidemic proportions of the mountain pine beetle (MPB) in British Columbia (BC), Canada. The result of this is increasing volumes of dead lodgepole pine with increasingly limited commercial uses. Bioconversion of lodgepole pine to ethanol using SO(2)-catalyzed steam explosion was investigated. The optimum pretreatment condition for this feedstock was determined to be 200 degrees C, 5 min, and 4% SO(2) (w/w). Simultaneous saccharification and fermentation (SSF) of this material provided an overall ethanol yield of 77% of the theoretical yield from raw material based on starting glucan, mannan, and galactan, which corresponds to 244 g ethanol/kg raw material within 30 h. Three conditions representing low (L), medium (M), and high (H) severity were also applied to healthy lodgepole pine. Although the M severity conditions of 200 degrees C, 5 min, and 4% SO(2) were sufficiently robust to pretreat healthy wood, the substrate produced from beetle-killed (BK) wood provided consistently higher ethanol yields after SSF than the other substrates tested. BK lodgepole pine appears to be an excellent candidate for efficient and productive bioconversion to ethanol.     

The phospholipid composition ofBradyrhizobium spp

The phospholipid composition of two strains ofBradyrhizobium is reported. In contrast to previous studies [Bunn CR, Elkan GH (1970) Can J Microbiol 17:291–295; and Gerson T, Patel JJ (1975) Appl Microbiol 30:193–198], we determined that phosphatidylglycerol is a major phospholipid within this bacterial genus. Furthermore, neither phosphatidylserine nor phosphatidylinositol was detected within lipid extracts derived from these bacteria. In addition to phosphatidylglycerol, other major phospholipids ofBradyrhizobium were shown to include phosphatidylcholine, phosphatidylethanolamine, and cardiolipin. Possible explanations for the discrepancies between the present study and those of previous investigations are discussed.     

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.     

Regulation of the tricarboxylic acid cycle in gram-positive, facultatively anaerobic bacilli.

The facultative anaerobes Bacillus polymyxa Hino G, B. polymyxa Hino J, and B.macerans were observed to have imcomplete tricarboxylic acid cycles. They were devoid of malate dehydrogenase and all had very low levels of alpha-ketoglutarate dehydrogenase. B. polymyxa Hino J was devoid of alpha-ketoglutarate dehydrogenase when grown aerobically and anerobically. Citrate synthase from B. polymyxa was inhibited by adenosine triphosphate but not reduced nicotinamide adenine dinucleotide and resembled enzymes from other gram-positive bacteria in this respect. Like the citrate synthases from gram-negative, facultative anaerobes and chemolithotrophs, the enzyme from B. polymyxa was inhibited by alpha-ketoglutarate. Inhibition by adenosine triphosphate was shown to be competitive with acetyl-coenzyme A and alpha-ketoglutarate inhibition was competitive with oxaloacetate.     

Comparison of the presence-absence and membrane filter techniques for coliform detection in small, nonchlorinated water distribution systems.

The traditional membrane filter (American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 16th ed., American Public Health Association, Washington, D.C., 1985) and presence-absence (P-A) (J. A. Clark, Can. J. Microbiol. 14:13-18, 1968) techniques for the detection of coliform bacteria were compared in a small nonchlorinated drinking water distribution system by using total positive samples and frequency-of-occurrence analyses. No significant differences (P less than 0.05) were found in detection of the presence of coliform bacteria or in changes in the frequency of occurrence with time. A reduction in P-A sample volume (to 50 ml) was not found to statistically affect the comparative results of traditional membrane filter and P-A tests.     

Comparison of the presence-absence and membrane filter techniques for coliform detection in small, nonchlorinated water distribution systems

The traditional membrane filter (American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 16th ed., American Public Health Association, Washington, D.C., 1985) and presence-absence (P-A) (J. A. Clark, Can. J. Microbiol. 14:13-18, 1968) techniques for the detection of coliform bacteria were compared in a small nonchlorinated drinking water distribution system by using total positive samples and frequency-of-occurrence analyses. No significant differences (P less than 0.05) were found in detection of the presence of coliform bacteria or in changes in the frequency of occurrence with time. A reduction in P-A sample volume (to 50 ml) was not found to statistically affect the comparative results of traditional membrane filter and P-A tests.