Phytoremediation of Soils Contaminated with Wood Preservatives: Greenhouse and Field Evaluations

Phytoremediation was evaluated as a potential treatment for the creosote-contaminated surface soil at the McCormick and Baxter (M&B) Superfund Site in Portland, Oregon. Soil at the M&B site is contaminated with pentachlorophenol (PCP) and polyaromatic hydrocarbons (PAHs). Eight individual PAH compounds (containing four to six aromatic rings) were included in the investigation. Greenhouse and field studies were carried out using perennial ryegrass (Lolium perenne). The following three treatments were compared in both studies: T1 = planted-amended soil; T2 = unplanted-amended soil; and T3 = unplanted-unamended soil. The amendments were mineral nutrients and dolomite, which was used to raise the acidic pH of the soil. Contaminant concentrations in the soil were measured initially and at regular intervals for several months. In the greenhouse study, the concentrations of certain contaminants decreased as a function of time. Thus, PCP, fluoranthene, pyrene, chrysene, and benzo(k)fluoranthene appeared to undergo biodegradation in all of the treatments. On the other hand, certain larger molecular weight PAHs were relatively recalcitrant. These “recalcitrant PAHs” included benzo(b)fluoranthene, benzo(a)pyrene, benzo(g,h,i)perylene and indeno(1,2,3-cd)pyrene. Statistical methods were used to compare the concentrations of the more easily biodegraded contaminants in treatments T1, T2, and T3. The statistical analysis was facilitated by normalizing the contaminant concentrations relative to the sum of the recalcitrant PAHs in the same sample. Thus, ratios were created that could be compared directly to benchmark values indicative of the contaminant at the beginning of the study. In the greenhouse study, statistically significant differences between T1 and T2, across all treatment times, were obtained for fluoranthene, pyrene, and chrysene (p ≤0.05), suggesting enhanced rhizosphere biodegradation for these compounds. Significant differences between T2 and T3 were obtained for pyrene (p ≤0.03), indicating that nutrients stimulated the biodegradation of this contaminant. Although the greenhouse study was carried out with a well-mixed soil sample from the M&B site, an extremely uneven distribution of contaminants was encountered in the field study. The resulting scatter in the field data made comparisons difficult, and treatment-specific effects observed in the greenhouse study were not statistically significant in the field study. However, analysis of the normalized data from the field revealed the same time-dependent decreases in contaminant concentration as observed in the greenhouse study.     

Feeding Preference of Native Subterranean Termites (Isoptera: Rhinotermitidae: Reticulitermes) for Wood Containing Bark Beetle Pheromones and Blue-Stain Fungi

Surprisingly little research has been conducted to investigate interactions between subterranean termites and bark beetles. Facilitative interactions between these organisms could easily alter stand dynamics and impact wood utilization strategies. A series of American Wood Protection Association Standard E1-09 “choice tests” were carried out to determine the feeding preference of Reticulitermes flavipes Kollar (Isoptera: Rhinotermitidae) for blue-stained sapwood and sapwood impregnated with various bark beetle pheromones. Reticulitermes flavipes exhibited a feeding preference for both air-dried and kiln-dried blue-stained sapwood, unstained sapwood treated with frontalin, and air-dried blue-stained sapwood treated with a 0.02% solution of both frontalin and endo-brevicomin. The implications of these results are far reaching, with particular relevance to forest health, ecology, and utilization.     

Effect of Temperature and Termite Starvation on Phagocytosis by Protozoan Symbionts of the Eastern Subterranean Termite Reticulitermes flavipes Kollar

Abstract Many termite species rely on intestinal protozoan symbionts to digest their cellulosic foods. We examined cellulose acquisition by the symbionts of the Eastern subterranean termite Reticulitermes flavipes Kollar (Isoptera; Rhinotermitidae) by following their phagocytosis of red paper fed to the termite host. The effects of termite host starvation and environmental temperature on feeding activity were studied in the zooflagellates Trichonympha agilis Leidy (Trichonymphidae), Pyrsonympha vertens Leidy, Dinenympha fimbriata Kirby, and D. gracilis Leidy (Pyrsonymphidae), which are among the largest residents in R. flavipes' hindguts. Protozoans in termites starved for 24 h ingested red paper significantly sooner than protozoans in termites with continuous access to food. Trichonympha, Pyrsonympha, and Dinenympha all ingested red paper particles at approximately the same rate. Red paper appeared significantly sooner in protozoans in termites maintained at 32°C than in those maintained at 22°C or 26°C. At 32°C, numbers of Trichonympha per gut remained constant over 96 h. Pyrsonympha and Dinenympha cells were absent or significantly reduced in number by 72 h at that temperature. These results provide insight into the environmental factors that shape the termite–protozoan symbiosis. They may aid in the development of protozoicides used to control pest termites. Received: 1 August 1997; Accepted: 26 November 1997     

Effect of Average Worker Size on Tunneling Behavior of Formosan Subterranean Termite Colonies

The effect of a colony's average worker size on tunneling behavior of Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) in laboratory foraging arenas was investigated. Large groups of foragers from four colonies were added to arenas and allowed to tunnel for a period of 22 days. Various aspects of the resulting tunnel galleries were analyzed using ANOVA and regression analysis. After the exclusion of one colony due to high mortality, the only parameters shown to differ significantly were tunnel width and tunnel segmentation. Larger workers tended to construct less segmented galleries with wider tunnels. Tunnel length was positively correlated to size on the first 2 days, but the relationship reversed by day 4. Tunneling on day 1 was positively correlated to the discovery of food items. Our results indicate that a foraging group's average worker size can affect tunnel pattern and that groups composed of larger workers are better tunnelers, but groups of smaller sized workers are able to make up the difference over time. Possible explanations for size-related variation in tunneling behavior are discussed.     

Premating Behavior of Dealates of the Formosan Subterranean Termite and Evidence for the Presence of a Contact Sex Pheromone

Swarming by alates of the Formosan subterranean termite, Coptotermes formosanus Shiraki, occurs around dusk during April–June in Louisiana and adjoining states. Dispersal is the primary purpose for swarming. We studied the premating behavior, following swarming. Loss of wings, especially in females, was essential for successful formation of tandem pairs between the two sexes. No calling was observed and apparently a long-range volatile sex pheromone, reported in some other species of termites, may not be involved in mate attraction in C. formosanus. Males moving randomly use their antennae to detect a female, and, together with maxillary and labial palps, contact is maintained with her while in tandem. Previously mated males paired with virgin females showed tandem behavior. The incidence was very low when both males and females were previously mated and then paired. Males with complete ablation of antennae did not form tandem pairs. Ablation of 10 terminal antennal segments or the labial palps caused a significant increase in the time to initiate tandem behavior. Electrophysiological recordings from gustatory sensilla on both antennae and maxillary palps revealed increased neural activity in response to female abdominal tip extracts compared to activity elicited by a solvent control. Analysis of extracts of male and female abdominal tips using high-performance liquid chromatography showed a peak unique to the female extract. We discuss the presence of a nonvolatile chemical in female C. formosanus and its involvement in the specific premating behavior.     

Escape Behavior of the Formosan Subterranean Termite (Isoptera: Rhinotermitidae) in Response to Disturbance

Disturbances to monitors and baits have often been noted as a confounding factor for the success of termite baiting systems. We studied escape behavior of Coptotermes formosanus Shiraki to a food source disturbance in the laboratory. Four peripheral chambers connected to the central food chamber through tubes served as escape routes from which escape patterns, escape and return times and differences in soldier and worker responses were recorded. We used two sand conditions: moist and dry to test whether favorable versus unfavorable conditions change escape behavior. The post disturbance responses were video recorded for 1 h. C. formosanus quickly escaped from the central chamber when disturbed. The time taken to reach the maximum number of termites that escaped ranged from 20 s to 210 s followed by a gradual return to the disturbed chamber. The majority of the escapees returned to the disturbed area within a few minutes after evacuation in both dry and wet sand conditions. A significantly higher percentage of termites escaped in wet sand arenas (43%) than in the dry sand arenas (31%). Our experiments demonstrate that C. formosanus is highly unlikely to abandon bait stations for extensive periods of time as a result of mechanical disturbances regardless of habitat quality.     

Evaluation of DNA Fingerprinting, Aggression Tests, and Morphometry as Tools for Colony Delineation of the Formosan Subterranean Termite

Multilocus DNA fingerprinting, aggression tests, and morphometry were compared to evaluate their potential for the delineation of colonies of Coptotermes formosanus Shiraki (Isoptera; Rhinotermitidae) in Hawaii. DNA fingerprinting segregates the termites from all collection sites and allows the assignment of all individuals to their original collection site. The genetic similarity of termites from different collection sites approaches the population's genetic background similarity, consequently collection sites represent independent colonies. Aggression between colonies is comparatively low and does not provide reliable colony delineation. Morphometry allows a 79% classification rate of termites to their colony of origin. No correlation among genetic similarities, aggression levels, and morphometric distances is found. Of the three investigated methods, we conclude that the genetic approach is the most useful tool for colony delineation in C. formosanus.     

A Kazal-Type Serine Protease Inhibitor from the Defense Gland Secretion of the Subterranean Termite Coptotermes formosanus Shiraki

Coptotermes formosanus is an imported, subterranean termite species with the largest economic impact in the United States. The frontal glands of the soldier caste termites comprising one third of the body mass, contain a secretion expelled through a foramen in defense. The small molecule composition of the frontal gland secretion is well-characterized, but the proteins remain to be identified. Herein is reported the structure and function of one of several proteins found in the termite defense gland secretion. TFP4 is a 6.9 kDa, non-classical group 1 Kazal-type serine protease inhibitor with activity towards chymotrypsin and elastase, but not trypsin. The 3-dimensional solution structure of TFP4 was solved with nuclear magnetic resonance spectroscopy, and represents the first structure from the taxonomic family, Rhinotermitidae. Based on the structure of TFP4, the protease inhibitor active loop (Cys⁸ to Cys¹⁶) was identified.     

Chemical Identification and Ethological Function of Soldier-Specific Secretion in Japanese Subterranean Termite Reticulitermes speratus (Rhinotermitidae)

We identified the soldier-specific compounds in the Japanese subterranean termite, Reticulitermes speratus, to clarify their ethological roles. Silica gel column chromatography separated one major soldier-specific compound in the hexane fraction accounting for 70–80% of the total amount of the fraction, while cuticular hydrocarbons constituted the rest. We identified the compound as β-selinene by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Comparative GC analyses of the major exocrine glands detected the compound in the soldier’s frontal gland. Both soldiers and workers made aggregation to the hexane fraction, as well as to the crushed heads and head extract of the soldiers. They did not aggregate to cuticular hydrocarbons, making it likely that β-selinene was the aggregation pheromone in this species. The opportunistic predator of this termite, Lasius japonicus, was also attracted to the compounds. The ant workers, therefore, would use the termite aggregation pheromone as a kairomone for hunting them.     

Queue Size Determines the Width of Tunnels in the Formosan Subterranean Termite (Isoptera: Rhinotermitidae)

We present a model of tunnel excavation by termites that requires no pheromone labeling of soil or work sites, but instead relies on tactile interactions and individuals who actively orient their movement. Potential termite excavators moved from the tunnel origin towards the distal end of the tunnel and formed a queue behind those termites at the digging face. Delayed termites excavated soil laterally from the tunnel wall at a position governed by their position in the queue of termites. By examining excavation under artificially induced conditions of longer and shorter queues of termites at the tunnel end, we showed that tunnel width increased with increased queue size and the rate of lateral excavation in a process we termed “digging pressure.”     

Effects of Caste on the Expression of Genes Associated with Septic Injury and Xenobiotic Exposure in the Formosan Subterranean Termite

As social insects, termites live in densely populated colonies with specialized castes under conditions conducive to microbial growth and transmission. Furthermore, termites are exposed to xenobiotics in soil and their lignocellulose diet. Therefore, termites are valuable models for studying gene expression involved in response to septic injury, immunity and detoxification in relation to caste membership. In this study, workers and soldiers of the Formosan subterranean termite, Coptotermes formosanus, were challenged by bacterial injection or by no-choice feeding with a sublethal concentration (0.5%) of phenobarbital. Constitutive and induced expression of six putative immune response genes (two encoding for lectin-like proteins, one for a ficolin-precursor, one for the Down syndrome cell adhesion molecule, one for a chitin binding protein, and one for the gram-negative binding protein 2) and four putative detoxification genes (two encoding for cytochrome P450s, one for glutathione S-transferase, and one for the multi antimicrobial extrusion protein), were measured via quantitative real time polymerase chain reaction and compared within and among 1) colonies, 2) treatment types and 3) castes via ANOVA. Eight genes were inducible by septic injury, feeding with phenobarbital or both. Colony origin had no effect on inducibility or differential gene expression. However, treatment type showed significant effects on the expression of the eight inducible genes. Caste effects on expression levels were significant in five of the eight inducible genes with constitutive and induced expression of most target genes being higher in workers than in soldiers.     

Trail Communication During Foraging and Recruitment in the Subterranean Termite Reticulitermes santonensis De Feytaud (Isoptera, Rhinotermitidae)

The search for food in the French subterranean termite Reticulitermes santonensis De Feytaud is organized in part by chemical trails laid with the secretion of their abdominal sternal gland. Trail-laying and -following behavior of R. santonensis was investigated in bioassays. During foraging for food termites walk slowly (on average, 2.3 mm/s) and lay a dotted trail by dabbing the abdomen at intervals on the ground. When food is discovered they return at a quick pace (on average, 8.9 mm/s) to the nest, laying a trail for recruiting nestmates to the food source. While laying this recruitment trail the workers drag the abdomen continuously on the ground. The recruitment trail is highly attractive: it is followed within a few seconds, by more nestmates, and at a quicker pace (on average, 6.4 mm/s) than foraging trails (on average, 2.9 mm/s). The difference between foraging and recruitment trails in R. santonensis could be attributed to different quantities of trail pheromone. A caste-specific difference in trail pheromone thresholds, with workers of R. santonensis being more sensitive to trails than soldiers, was also documented: soldiers respond only to trails with a high concentration of trail pheromone.     

Infection of the Eastern Subterranean Termite,Reticulitermes flavipes (Kollar) with the fungusBeauveria bassiana (Balsamo) Vuill.

Observations were made on the histopathology of the eastern subterranean termite,Reticulitermes flavipes (Kollar) infected with the fungusBeauveria bassiana (Balsamo) Vuill. Initial symptoms of infected termites were sluggishness, anal discharge, passivity, and failure to respond to tactile stimuli. Infected fifth instar worker termites changed from a healthy creamy color to a light brown. Penetration of the integument by germ tubes ofB. bassiana occurred as early as 16 hours after inoculation. Infection through the digestive tract also resulted. Following penetration of the integument the fungus attacked the fat body, then the nervous tissue and finally the musculature.     

Field performance in northern Spain of Douglas-fir seedlings Inoculated with ectomycorrhizal fungi

 Experimental plantations were established in northern Spain to determine the effects of different ectomycorrhizal fungi on growth and survival of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) under field conditions. Douglas-fir seedlings were inoculated with Laccaria bicolor S238 mycelia in two bareroot nurseries in central France or with spore suspensions of three hypogeous ectomycorrhizal species: Melanogaster ambiguus, Rhizopogon colossus and R. subareolatus, in a Spanish containerised nursery. The effects of ectomycorrhizal inoculation on plant survival after outplanting were limited, being only significant at the Guipuzkoan (Spain) site, when plants inoculated with L. bicolor S238 were compared to non-inoculated plants grown in non-fumigated soil. L. bicolor S238 had a significant effect on plant growth during the phase of bareroot nursery growth and this difference was maintained after field outplanting. Nursery inoculations with M. ambiguus, R. colossus and R. subareolatus improved plant growth during the first 2 and 3 years after field outplanting. The positive effects of the inoculation treatment on seedling height, root collar diameter and stem volume persisted after 5 years of field growth. Inoculation with these ectomycorrhizal fungi may improve the field performance of Douglas-fir seedlings in northern Spain. Accepted: 12 February 1999     

RSPCA Mallydams Wood and Field Study Centre

Recent research into the ability of plants to detect near neighbours is reviewed. The detection mechanism is believed to be through the phytochrome equilibrium. A simple experiment with radishes grown near a wheat hedge is reported. Plants growing near the hedge directed more of their growth into leaves. As distance from the hedge increased, so did the proportion of photosynthates directed into storage roots. This effect was noticeable up to about a metre from the hedge. The ecological applications of this for plants growing in the natural environment can be seen everywhere.     

Fungi Isolated from Flue-Cured Tobacco Inoculated in the Field with Storage Fungi

Flue-cured tobacco inoculated in the field with A. amstelodami, A. flavus, A. ochraceus, A. repens, A. ruber, and a species of Penicillium was rarely invaded by these fungi. Regardless of inoculum, the predominant fungi reisolated from green tissue were species of Alternaria and Cladosporium. After curing, A. repens, A. niger, and species of Alternaria and a species of Penicillium were the most commonly isolated fungi. The fungus used as inoculum was not the predominant fungus reisolated from green or cured tissue. Conditions during handling and storage prior to marketing probably determine when storage fungi become associated with the leaf and which species becomes predominant.     

Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.     

Long-term Field Efficacy of the Entomogenous Fungus Metarhizium anisopliae against the Subterranean Scarab, Adoryphorus couloni

The efficacy of Metarhizium anisopliae DAT F-001 against Adoryphorus couloni was examined over 4 years on a sheep grazing property ('Inverell') in central Tasmania. Three generations of the primary A. couloni population and two generations of the overlapping population were studied. Application of 5.1 0.7 104 M. anisopliae spores g-1 of soil (2 cm below the soil surface), during midwinter 1989, when the primary population was in the middle of the L3 larval stage, resulted in 30.3% fewer larvae in treated plots by 21 weeks and 57.8% less pupae by 27 weeks. The population decline was consistent with a mortality model developed from laboratory data. In the two subsequent generations of the primary population, there were 63.2% (1991) and 45.0% (1993) fewer larvae in the treated plots before the damaging L3 stage. The two sequential generations of the overlapping A. couloni larval populations had 68% (1990) and 65% (1992) fewer larvae in the treated plots than in the untreated plots. Reductions in larval numbers led to a greater retention of sown perennial grasses, reduced weed invasion and a 23% increase in pasture productivity in the autumn of 1992. Incorporation of M. anisopliae into the soil did not reduce the numbers of non-target invertebrates. The level of M. anisopliae DAT F-001 in the pasture remained at levels close to the applied concentration (5.1 0.7 colony-forming units g-1 of soil), but increased 10-fold when mummified L3 larval, prepupal and pupal cadavers were present from January to June 1990. The level of the fungus in the soil was still twice the original applied concentration at the conclusion of sampling in March 1992.