The cutworm Peridroma saucia (Lepidoptera: Noctuidae) supports growth and transport of pBR322-bearing bacteria.

Variegated cutworms were exposed to bean plants in microcosms sprayed with pBR322-carrying strains of Enterobacter cloacae, Klebsiella planticola, and Erwinia herbicola. The three bacterial species exhibited differential survival on leaves, in soil, and in guts and fecal pellets (frass) of the insects. High numbers of Enterobacter cloacae(pBR322) were detected in all samples, while the other species were unable to establish residence in the insect. To assess the impact of this colonization on site-to-site transport of microorganisms, larvae were fed plants that had been sprayed with the bacteria and then were transferred to uninoculated plants. Cutworms were efficient carriers of Enterobacter cloacae(pBR322), as indicated by its rapid appearance on uninoculated leaves and continued persistence in the insects for 3 days after transfer. Few Erwinia herbicola(pBR322) and K. planticola(pBR322) were obtained from larvae after transfer, although up to 10(3) CFU/g were detected in soil and on plants. Differences in bacterial survival and growth were confirmed by incubating frass overnight and observing the change in population numbers. The proportion of total samples showing at least a 25-fold increase during incubation was 68% for Enterobacter cloacae(pBR322), 39% for K. planticola(pBR322), and 0% for Erwinia herbicola(pBR322). Our results emphasize the role that cutworms and possibly other insects have in persistence and growth of microorganisms in the environment.     

Survival of bacteria during aerosolization

One form of commercial application of microorganisms, including genetically engineered microorganisms is as an aerosol. To study the effect of aerosol-induced stress on bacterial survival, nonrecombinant spontaneous antibiotic-resistant mutants of four organisms, Enterobacter cloacae, Erwinia herbicola, Klebsiella planticola, and Pseudomonas syringae, were sprayed in separate experiments in a greenhouse. Samples were collected over a distance of 15 m from the spray site for enumeration. Spores of Bacillus subtilis were used as tracers to estimate the effects of dilution on changes in population over distance. Viable counts of P. syringae, Enterobacter cloacae, and K. planticola decreased significantly over a distance of 15 m. Erwinia herbicola showed no significant decline in counts over the same distance. The degree of survival of P. syringae during aerosolization was dependent on ambient environmental conditions (i.e., temperature, relative humidity), droplet size of the aerosol, and prior preparative conditions. Survival was greatest at high relative humidities (70 to 80%) and low temperatures (12 degrees C). Survival was reduced when small droplet sizes were used. The process of washing the cells prior to aerosolization also caused a reduction in their survival. Results from these experiments will be useful in developing sound methodologies to optimize enumeration and for predicting the downwind dispersal of airborne microorganisms, including genetically engineered microorganisms.     

Survival of bacteria during aerosolization.

One form of commercial application of microorganisms, including genetically engineered microorganisms is as an aerosol. To study the effect of aerosol-induced stress on bacterial survival, nonrecombinant spontaneous antibiotic-resistant mutants of four organisms, Enterobacter cloacae, Erwinia herbicola, Klebsiella planticola, and Pseudomonas syringae, were sprayed in separate experiments in a greenhouse. Samples were collected over a distance of 15 m from the spray site for enumeration. Spores of Bacillus subtilis were used as tracers to estimate the effects of dilution on changes in population over distance. Viable counts of P. syringae, Enterobacter cloacae, and K. planticola decreased significantly over a distance of 15 m. Erwinia herbicola showed no significant decline in counts over the same distance. The degree of survival of P. syringae during aerosolization was dependent on ambient environmental conditions (i.e., temperature, relative humidity), droplet size of the aerosol, and prior preparative conditions. Survival was greatest at high relative humidities (70 to 80%) and low temperatures (12 degrees C). Survival was reduced when small droplet sizes were used. The process of washing the cells prior to aerosolization also caused a reduction in their survival. Results from these experiments will be useful in developing sound methodologies to optimize enumeration and for predicting the downwind dispersal of airborne microorganisms, including genetically engineered microorganisms.     

Microbial mediation of fruit fly–host plant interactions: is the host plant the “centre of activity”?

Insects utilize resources in their environment with the aid of mutualistic or symbiotic mediation by microorganisms. Some insect species such as ants and termites often have complex ecological and evolutionary associations with their symbionts, while the nature and functional significance of such associations in non-social insects is often unclear. In the Dacinae (Diptera: Tephritidae), specific Enterobacteriaceae ( Erwinia herbicola , Enterobacter cloacae , Klebsiella oxytoca ) are believed to mediate interactions between the adult fruit flies and the larval host plant. This bacterial mediation is hypothesized as being integral to the larval host plant being the "centre of activity" of the fly. Using a non-pest, monophagous fruit fly ( Bactrocera cacuminata [Hering]), we tested this hypothesis by manipulating the fruiting state of its larval host plant ( Solanum mauritianum Scopoli) and subsequently assessing insect behaviour and phylloplane microflora on those hosts. On host plants that had never fruited, few flies or bacterial colonies were recorded, consistent with hypothesis expectations. On fruiting host plants or plants that had had their fruit removed, bacterial colonies were present; again consistent with expectation. However, few flies were recorded on fruit-removed plants and all fly behaviours, other than resting or oviposition, were rare or absent on any hosts; inconsistent with expectation. The general pattern of results suggested that female flies coming to oviposit on fruiting hosts were spreading Enterobacteriaceae, but such spread was incidental and not part of some mutualistic interaction between fruit flies and bacteria.     

Biological control of an insect pest by gut-colonizing Enterobacter cloacae transformed with ice nucleation gene

The ice nucleation (IN) gene inaA of epiphytic Erwinia (Pantoea) ananas IN10 was transformed into Enterobacter cloacae WBMH-3-CMr originated from the faeces of silkworms. The transformant designated as Ent. cloacae WBMH-3-CMr(pICE6S13) exhibited IN activity, unlike the parent strain. The transgenic strain was ingested by mulberry pyralid larvae, fed on detached mulberry leaves, and the supercooling capacity and cold hardiness of these larvae were examined. The mean supercooling point (SCP) of the larvae ingesting the transgenic strain was - 3.3 degrees C, 8 degrees C higher than that of larvae treated with distilled water (control) and 1.5 C higher than an ice nucleation active (INA) strain of Erw. ananas. The SCPs of the larvae were stably maintained over the 9 d after ingestion. The maintenance of these high SCPs was due to transgenic Ent. cloacae having a more stable and efficient gut colonization than Erw. ananas, which is identified by the distribution of a narrower range of SCPs (-2 to -5 degrees C) in larvae treated with the transgenic stain. Furthermore, most of the larvae ingesting the transgenic strain froze and died when they were exposed to cold conditions of -5 degrees C for 18 h, 3 or 7 d after ingestion. In contrast, most of the larvae ingesting no bacterium did not die under similar conditions. On the other hand, the growth ability of Ent. cloacae WBMH-3-CMr on mulberry leaves tended to be lower than that of epiphytic Erw. ananas, as assayed by pot tests. These findings would expand the possibility of biological control using INA bacteria since Ent. cloacae would harbour a broader host (insect) range for gut colonization and a smaller affinity to plants to benefit from prevention of plant frost injury.     

The Ecology of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> on the Phylloplane: Colonization from Soil,Plasmid Transfer,and Interaction with Larvae of <Emphasis Type="Italic">Pieris brassicae</Emphasis>

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.     

Transgenic ice nucleation-active Enterobacter cloacae reduces cold hardiness of corn borer and cotton bollworm larvae

The ice nucleation (IN) gene iceA of Erwinia ananas 110 was integrated into the chromosomes of two Enterobacter cloacae strains (Enc1.2022 and Enc1.181). These two newly derived transgenic strains, designated Enc2022-I and Enc181-I, respectively, possessed ice nucleation activity at -2.5 degrees C, significantly higher than their parent strains (active at approx -10 degrees C or lower). After ingesting these transgenic bacteria, the mean supercooling points (SCPs) of corn borer and cotton bollworm larvae were -3 to -4 degrees C, significantly higher than those of untreated controls. The SCPs remained significantly elevated over the 9-day period after ingestion, which matched well with the efficient gut colonization of the bacteria during this period. All treated larvae froze and eventually died after exposure for 6 h to a temperature of -7 degrees C, and more than 95% died after 12 h at -5 degrees C. In contrast, few or none of the untreated control larvae froze and died under the same conditions. Furthermore, the growth ability of these transgenic ice nucleation-active (INA) En. cloacae strains on corn leaves was reduced, compared to that of wild-type epiphytic E. ananas, as revealed by pot tests conducted in both greenhouse and outdoor conditions. The stable colonization in insect guts and their lower affinity to plants would make these transgenic INA bacteria useful as a novel tool for biological control of insect pests in agricultural fields.     

Nitrogen fixation associated with 'Park' Kentucky bluegrass (Poa pratensis L.).

Associative nitrogen fixation in Kentucky bluegrass (Poa pratensis L.) turfs inoculated with five nitrogen-fixing bacterial isolates was evaluated using the acetylene reduction assay and nitrogen accumulation as indicators of fixation. 'Park' and 'Nugget' Kentucky bluegrass turfs were grown in controlled environment chambers and inoculated with Klebsiella pneumoniae (W-2, W-6, and W-14), Erwinia herbicola (W-8), and Enterobacter cloacae (W-11). 'Park' inoculated with K. pneumoniae (W-6) had significant acetylene reduction activity using undisturbed turfs. Other treatments including turfs treated with heat-killed cells had no significant difference in acetylene reduction. In a second study, "Park' and 'South Dakota Certified' turfs were grown in a greenhouse and inoculated with K. pneumoniae (W-6) and E. herbicola (W-8). 'Park' inoculated with K. pneumoniae (W-6) had increased acetylene reduction activity rates and also a greater nitrogen accumulation in aerial tissues when compared to controls. Acetylene reduction activity was correlated (r = 0.92) to nitrogen accumulation. Other treatments did not effectively increase acetylene reduction activity or nitrogen accumulation.     

Positive and negative impacts of insect frass quality on soil nitrogen availability and plant growth

Frass deposition to soil is an important pathway by which herbivorous insects impact decomposition and soil nutrient availability. However, little is known about how frass quality influences ecosystem properties. Here, we examined the effects of frass quality on the decomposition process, soil nitrogen (N) availability, and plant growth, using frass of Mamestra brassicae (L.) that fed on fertilized or unfertilized Brassica rapa L. var. perviridis Bailey. The frass quality was largely dependent on the host plant quality. Frass excreted by larvae that fed on the fertilized plants had higher N than that of larvae that fed on the unfertilized plants. The decomposition rate of the frass did not differ between N-rich and N-poor frass, except during the early decomposition period. The inorganic N concentration decreased during decomposition in both frass types. However, difference in the initial inorganic N concentration led to different consequences regarding soil N availability. Furthermore, addition of frass to the soil differently influenced the growth of B. rapa plants depending on the frass quality: plant biomass was increased by N-rich frass addition but decreased by N-poor frass addition, compared to the biomass without frass addition. These results indicate that frass quality is an important factor in determining the impact of herbivorous insects on nutrient dynamics, and that frass positively or negatively influences soil N availability and plant growth, depending on its quality.     

Effect of Bacillus thuringiensis naturally colonising Brassica campestris var. chinensis leaves on neonate larvae of Pieris brassicae

The feeding of neonate larvae of Pieris brassicae (Order Lepidoptera) on leaves of brassica plants that had been colonised by Bacillus thuringiensis resulted in the death of 35% of the population within 72 h. The bacteria multiplied in the cadavers, resulting in an increase of about 50-fold compared to the living insects. Surviving insects showed no ill effects during the time of the study. There was negligible multiplication of B. thuringiensis in the frass.     

Erwinia aphidicola,a new species isolated from pea aphid,Acyrthosiphon pisum

Five strains of Gram-negative, oxidase-negative, facultatively anaerobic, fermentative, motile, rod-shaped bacterium with the general characteristics of the family Enterobacteriaceae were isolated from the gut of multiple specimens of the pea aphid. All the strains caused aphid mortality when ingested by insects via a synthetic diet. The results of biochemical tests showed that these strains are most related to Erwinia herbicola and Pantoea agglomerans. According to DNA-DNA hybridization, the five strains showed more than 96% relatedness to each other, indicating that these organisms are members of a single species. These strains were most closely related to Erwinia herbicola (22% DNA relatedness). Phenotypic differentiation of these strains from Erwinia herbicola, which was also detected from aphid gut, was based on negative reactions in tests of yellow pigment production, gelatin liquefaction, acid production from inulin, starch and dulcitol, and positive acid production from melibiose, inositol, cellobiose and glycerol. On the basis of these data, the name Erwinia aphidicola is proposed for the new organism. The type strain is strain X 001 (=IAM 14479).     

Immune responses in Rhodnius prolixus: influence of nutrition and ecdysone

Starved larvae of Rhodnius prolixus, when challenged with Enterobacter cloacae B12, had their mortality related to their period of starvation. R. prolixus larvae fed on plasma alone, compared with insects fed on whole blood, had their immune reactivity affected as shown by: (i) a significant reduction in the ability to produce cecropin-like and lysozyme activities in the haemolymph when inoculated with E. cloacae; (ii) a reduction in numbers of haemocytes and nodule formation following challenge with bacteria; (iii) a decreased ability of plasma-fed insects in destroying their infection caused by inoculation of E. cloacae cells; and (iv) alpha-ecdysone therapy counteracted the immune depression in Rhodnius larvae fed on plasma alone. However, unlike other immune reactions, this set of experiments failed to demonstrate any interference of the plasma feeding on the prophenoloxidase-activating system, since melanin production was not reduced when the system was stimulated by the presence of bacteria in the haemolymph. The significance of these data is discussed in relation to the effect of diet components and the moulting hormone on the immune reactivity in insects.     

Effect of aerosolization on subsequent bacterial survival

To determine whether aerosolization could impair bacterial survival, Pseudomonas syringae and Erwinia herbicola were aerosolized in a greenhouse, the aerosol was sampled at various distances from the site of release by using all-glass impingers, and bacterial survival was followed in the impingers for 6 h. Bacterial survival subsequent to aerosolization of P. syringae and E. herbicola was not impaired 1 m from the site of release. P. syringae aerosolized at 3 to 15 m from the site of release at a temperature of 12 degrees C and a relative humidity of 80% survived 35- to 65-fold better than P. syringae released at 27 degrees C and a relative humidity of 40%. No difference was observed in the survival of P. syringae and E. herbicola following aerosolization at the same temperature and relative humidity. Bacteria sprayed directly onto bean and oat plants established stable populations at comparable numbers on both plants over an 8-day period following inoculation. Bacteria that inoculated adjacent plants by drifting downwind up to 5 m were detectable at an initial population of 10(2) CFU/g on oats and 10(5) CFU/g on beans 2 h after the spray. However, bacterial populations on both plants were undetectable within 48 h.     

Distribution and residual activity of two insecticidal proteins, avidin and aprotinin, expressed in transgenic tobacco plants, in the bodies and frass of Spodoptera litura larvae following feeding

To understand how a major cosmopolitan pest responds to two very different insecticidal proteins and to determine whether herbivorous insects and their frass could be environmental sources of recombinant proteins from transgenic plants, Spodoptera litura (Fab.) (Lepidoptera, Noctuidae) larvae were fed on tobacco leaves expressing either the biotin-binding protein, avidin, or the protease inhibitor, aprotinin. Control larvae received non-transgenic tobacco. Samples of larvae were taken after 5, 6 or 7 days’ feeding and frass was collected after two 24-h periods at 6 and 7 days. Insects in all treatments grew significantly during the experiment, but the avidin-fed larvae were significantly smaller than the others on Day 7. Avidin was found in all samples of avidin-fed larvae (7.0±0.86 ng mg⁻¹, n=45), at a lower level than in their frass (31.9±5.08 ng mg⁻¹, n=30), and these frass levels were lower than those of the the leaves fed to the larvae (69.0±6.71 ng mg⁻¹, n=45). All of the avidin detected in these samples was capable of binding biotin. On average, between 10 and 28% of avidin was recovered with the methods used, whereas almost full recovery of aprotinin was effected. Aprotinin levels in larvae (8.2±0.53 ng mg⁻¹, n=45) were also lower than aprotinin levels in frass (77.4±6.9 ng mg⁻¹, n=30), which were somewhat lower than those in the leaves fed to the larvae (88.6±2.51 ng mg⁻¹, n=45). Approximately half the trypsin-binding ability of aprotinin was lost in larvae, and in frass, aprotinin had lost about 90% of its ability to bind trypsin.     

Effect of solar radiation and predacious microorganisms on survival of fecal and other bacteria.

The effect of solar radiation and predacious microorganisms on the survival of bacteria of fecal and plant origin was studied. The decline in the numbers of Escherichia coli cells in estuarine water samples was found to be significantly greater in the presence of both naturally occurring microbial predators and solar radiation than when each of these factors was acting independently. The effect of solar radiation on microbial predators was negligible, whereas the susceptibility of bacteria to light-induced decay varied from one organism to another, as follows: Klebsiella pneumoniae greater than E. coli greater than Salmonella typhimurium, Streptococcus faecium, Enterobacter aerogenes, Erwinia herbicola.     

Impact of Spodoptera littoralis (Boisd.) and Agrotis ipsilon (Hufn.) larval frass on oviposition of conspecific insects

Ethanolic extracts of larval frass of the cotton leaf worm and the black cutworm were prepared and tested to deter the eggs lay of the adults of the same insects. Two different types of food were used for larval feeding. Extracts fractions were identified using gas chromatography–mass spectrometry analysis. High concentrations were more deterrent to oviposition than low. Extracted frass of fed larvae on semi-artificial diet was more effective than others fed on natural host. Sensitivity of the black cutworm adult females to the cotton leaf worm frass extract was clearly observed at high tested concentration of L_1–3 and L₄ frass extracts which resulted completely oviposition deterrent. Several fatty acids were identified qualitatively and quantitatively in frass extracts of different larval instars of both target insects. Type and quantity of fatty acid depends mainly on larval food source and larval instar, except palmitic acid which recorded at all larval instars and food sources. Oleic acid and ethyl 9-hexadecaenoic acid were found when semi-artificial diet used as a food source while myrisitic acid was observed only in extracted frass of fed larvae on castor oil leaves.     

Effect of aerosolization on subsequent bacterial survival.

To determine whether aerosolization could impair bacterial survival, Pseudomonas syringae and Erwinia herbicola were aerosolized in a greenhouse, the aerosol was sampled at various distances from the site of release by using all-glass impingers, and bacterial survival was followed in the impingers for 6 h. Bacterial survival subsequent to aerosolization of P. syringae and E. herbicola was not impaired 1 m from the site of release. P. syringae aerosolized at 3 to 15 m from the site of release at a temperature of 12 degrees C and a relative humidity of 80% survived 35- to 65-fold better than P. syringae released at 27 degrees C and a relative humidity of 40%. No difference was observed in the survival of P. syringae and E. herbicola following aerosolization at the same temperature and relative humidity. Bacteria sprayed directly onto bean and oat plants established stable populations at comparable numbers on both plants over an 8-day period following inoculation. Bacteria that inoculated adjacent plants by drifting downwind up to 5 m were detectable at an initial population of 10(2) CFU/g on oats and 10(5) CFU/g on beans 2 h after the spray. However, bacterial populations on both plants were undetectable within 48 h.     

Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.

Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within pyruvate decarboxylases of various fungal and plant species also exhibited considerable homology to portions of this gene. This gene therefore presumably encodes an indolepyruvate decarboxylase (IpdC) which catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. Insertions of Tn3-spice within ipdC abolished the ability of strain 299R to synthesize indole-3-acetaldehyde and tryptophol and reduced its IAA production in tryptophan-supplemented minimal medium by approximately 10-fold, thus providing genetic evidence for the role of the indolepyruvate pathway in IAA synthesis in this strain. An ipdC probe hybridized strongly with the genomic DNA of all E. herbicola strains tested in Southern hybridization studies, suggesting that the indolepyruvate pathway is common in this species. Maximum parsimony analysis revealed that the ipdC gene is highly conserved within this group and that strains of diverse geographic origin were very similar with respect to ipdC.     

Antagonistic Activities of Epiphytic Bacteria from Soybean Leaves against Pseudomonas syringae pv. glycinea in vitro and in planta

Abstract Over two growing seasons, 273 bacterial strains were isolated from soybean leaves without and with bacterial blight symptoms caused by Pseudomonas syringae pv. glycinea (Psg). The majority of the isolates from leaves with symptoms were identified as Psg (43%), followed by Erwinia herbicola (21%), and Enterobacter/Erwinia (19%). The isolates from leaves without symptoms included mainly a group of unidentified Gram-negative bacteria (22%), Psg (21%), and E. herbicola (18%). Psg colonized the soybean leaves prior to saprophytic bacteria, and remained dominant during both seasons on healthy, as well as infected, leaves. Eighty-two saprophytic isolates were tested in vitro for their antagonistic activities against Psg, using an agar-diffusion assay. For the in planta assay, Psg and each isolate were simultaneously inoculated into wounds of pin-pricked leaves of greenhouse-grown soybean plants. Twenty-nine isolates were antagonistic in vitro. Nineteen isolates were able to suppress the growth of Psg and prevented the formation of leaf spots in planta when mixtures of isolate and pathogen were inoculated at ratios >1. Only 9 of the 82 isolates inhibited Psg in vitro as well as in planta. Most antagonists detected belonged to the genera Pseudomonas and the species Erwinia herbicola. The in planta assay should be a reliable predictor of field performance for screening of biological control agents. Received: 8 April 1996; Accepted: 22 October 1996