Computer tracking as a behavioral GC detector: Nematode responses to vapor of host roots

A computer tracking system previously developed for studying the behavioral responses of nematodes to volatile stimuli was modified so that the effluent of a gas chromatograph passed over the nematodes while their behavior was analyzed. Since the behavior of several hundred nematodes can be analyzed simultaneously, this technique has the potential to provide a sensitive assay for behaviorally active fractions from the GC. This potential was tested with infective juveniles of the root-knot nematodeMeloidogyne incognita and vapor from the roots of host tomato plants. Direct injection of 1 ml of vapor led to reproducible responses at times corresponding to nitrogen plus oxygen elution and to carbon dioxide elution. Experiments with pure gases demonstrated responses to oxygen and to carbon dioxide. Attempts were made to find less volatile and less active components by pulling large volumes of vapor through a trap of Tenax GC and subsequently eluting them onto the GC. This approach did not lead to the detection of other activities. We conclude that, other than carbon dioxide, there are no volatile stimuli released from host roots in effective quantities.     

Suppression of plant parasitic nematodes in the chinampa agricultural soils

Soil from the chinampa agricultural system in the Valley of Mexico suppressed damage by plant-parasitic nematodes to tomatoes and beans in greenhouse and growth chamber trials. Sterilization of the chinampa soil resulted in a loss of the suppressive effect, thereby indicating that one or more biotic factors were responsible for the low incidence of nematode damage. Nine organisms were isolated from chinampa soil, which showed antinematodal properties in culture. Naturally occurring populations of plant-parasitic nematodes were of lower incidence in chinampa soil than in Chapingo soil.     

Quantitative bioassay for chemotaxis with plant parasitic nematodes

A simple, flexible, and quantifiable bioassay for the attraction or repulsion of plant parasitic nematodes to or from root fractions or pure substances is described. Accurate gradients of volatile and nonvolatile substances can be measured and established. The method entails placing the nematodes in narrow agarose tracks such that their movement is essentially linear and the distance a population has traveled away or toward a given substance can be monitored with time. Plastic plates, each containing 10 such tracks, are described. The method is illustrated with second-stage larvae ofMeloidogyne incognita and a volatile attractant and nonvolatile repellent fraction obtained from cucumber roots.     

Sorgoleone from root exudate inhibits mitochondrial functions

The aim of this investigation was to determine if sorgoleone (SGL), a hydrophobic compound inSorghum bicolor (L.) Moench root exudate, interferes with mitochondrial functions. Tests were conducted on mitochondria isolated from etiolated soybean [Glycine max (L.) Merr.] and corn (Zea mays L.) seedlings. The data show SGL is a potent inhibitor of state 3 and state 4 respiration rates in both soybean and corn. Using either NADH, succinate, or malate as substrate, the I₅₀ was about 0.5M SGL for state 3 and 5.0M for state 4 based on 0.3–0.5 mg mitochondrial protein. Absorption spectra indicate SGL blocks electron transport at theb-c ₁ complex. These data show that disruption of mitochondrial function may be a mechanism of SGL-mediated growth inhibition previously reported and demonstrate a probable role of SGL inSorghum allelopathy.This study was supported in part by National Science Foundation grant RII-8902066 (South Dakota EPSCOR).     

Responses of plant-parasitic nematodeMeloidogyne incognita to carbon dioxide determined by video camera-computer tracking

A computer tracking system was used to quantify the responses of infective second-stage juveniles of the plant-parasitic nematodeMeloidogyne incognita to carbon dioxide. A sudden increase in concentration caused an increase in the rate of locomotion and a decrease in the frequency of changes of direction. The threshold was about 0.01 % vol CO₂/vol gas when the baseline concentration was very low and 0.05% CO₂ when the baseline concentration was 1% CO₂. The latter value represents a relative change of 5%. Concentrations above 10% CO₂ caused a general decrease in movement. In a second type of experiment, a constant concentration gradient of CO₂ was established, and the net movement of the nematodes along the gradient was determined. At low concentrations, the threshold was about 0.02% CO₂/cm. At higher concentrations, the threshold gradient was below 0.01% CO₂/cm or a relative gradient of less than 1% change/cm. At all concentrations to which nematodes responded they were attracted. The degree of orientation was estimated to be approximately 10% under most conditions. The rate of migration under the most favorable conditions was about 0.7 cm/hr. Three possible functions of the response are discussed: attraction to roots, movement toward optimal depth in soil, and as a collimating stimulus.     

Strong repellency of the root knot nematode,Meloidogyne incognita by specific inorganic ions

Simple inorganic salts of the ions K⁺, NH ₄ ⁺ , Cs⁺, NO ₃ ^– , and Cl^– are strongly repellent to infective second-stage larvae of the root knot nematode,Meloidogyne incognita. Some of these salts are known to be beneficial to plant growth. The results suggest a new means of plant protection.     

Repellent properties of the host compound 4-allylanisole to the southern pine beetle

The phenylpropanoid 4-allylanisole is a compound produced by loblolly pines (Pinus taeda L.), an abundant species in southern pine forests and a preferred host of southern pine beetle (Dendroctonus frontalis Zimmermann). Repellency of individual beetles was demonstrated in laboratory behavioral assays ofD. frontalis and other scolytids. Inhibition was demonstrated in natural populations ofD. frontalis using baited traps. In both tests, response to the inhibitory pheromone verbenone was used for comparison. In the laboratory, a higher proportion of newly emerged and reemergedD. frontalis responded negatively to 4-allylanisole than to verbenone. However, fewer reemergent than newly emerged individuals responded to either compound. In all field trials, the response ofD. frontalis to its attractant pheromone in funnel traps was significantly reduced by simultaneous release of 4-allylanisole. In most trials total reduction did not differ from verbenone; however, unlike verbenone, 4-allylanisole reduced male and female catches proportionally. Both compounds together did not significantly further reduce trap catch. The response of a major predator,Thanasimus dubius (F.), to the attractant pheromone ofD. frontalis, did not differ with the simultaneous release of either verbenone or 4-allylanisole. The results of preliminary field applications are presented and discussed.The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service.     

Theoretical range over which bacteria and nematodes locate plant roots using carbon dioxide

A theory used to estimate the smallest relative chemical gradient that is potentially detectable is given approximately byG>(DCR ²t³)^–1/2 whereD is the diffusion constant andC the concentration of the stimulus chemical,R is the effective radius of the receptor, the velocity of the organism, andt the time period within which the organism measures the concentration. Of these factors, time has the strongest influence. Combining this result with available information on gradients of carbon dioxide around roots and behavior of bacteria and nematodes leads to several conclusions. Bacteria can potentially detect carbon dioxide gradients in the layer of water in contact with the root but not further away. In contrast, plant-parasitic nematodes can potentially detect gradients one meter from a single long root fiber and over 2 m from a plant root mass using klinokinesis. A direct approach using klinotaxis can start a few centimeters from a single root fiber and half a meter from a root mass. These differences are due to differences in the time available to measure the concentrations. Increasing the length of a bacterium could increase its ability to detect gradients by reducing its rotational diffusion. Collimating stimuli that serve to maintain a straight path may provide a means of improving chemotaxis by permitting concentration to be measured for longer times during klinokinesis. More accurate predictions can be made when more precise data are available. The analysis is applicable to a wide variety of other organisms and stimuli.     

Control of plant-parasitic nematodes by a nematicidal strain ofAspergillus niger

An isolate ofAspergillus niger (designated PD-42) was evaluated in laboratory, greenhouse, and field trials for efficacy in controlling plant-parasitic nematodes. In greenhouse experiments, PD-42 drenches containing spores of PD-42 on oatmeal significantly reduced galling on tomato due to root-knot nematode as compared to untreated controls. In a one-half acre field experiment, PD-42 incorporated in seed coats was associated with significantly increased yield and decreased root-knot galling on pepper. In a second one-half acre field experiment, PD-42 drenches significantly reduced tomato and pepper root galling due toMeloidogyne incognita, and nonsignificant yield increases occurred. In each field experiment, treatment with PD-42 reducedRotylenchulus reniformis populations. The nematicidal components of theA. niger culture filtrates include citric acid, oxalic acid, and undetermined molecules larger than 8000 MW.     

Characterization of Dog Repellent Factor From Cuticular Secretion of Female Yellow Dog Tick, Haemaphysalis leachi

During its natural life cycle, the yellow dog tick, Haemaphysalis leachi, has three hosts, and it has to spend enough time on each of them to complete a blood meal. When irritated, the females of this tick species produce a cuticular secretion that contains a dog-repelling allomone. This improves the tick's chances of survival by deterring the dog from biting the tick off its body. Employing response-guided isolation techniques in conjunction with gas chromatography-mass spectrometry, the defensive allomone of H. leachi was found to consist of the six homologous aliphatic aldehydes from hexanal to undecanal. A mixture of synthetic versions of these six aldehydes in quantities corresponding to those secreted by one tick elicited strong aversion reactions in the majority of dogs of various breeds.     

α-Tomatine and resistance of tomato cultivars toward the nematode,Meloidogyne incognita

Susceptible and resistant tomato cultivars were assayed for root tomatine concentrations at different developmental times. No correlation between high tomatine levels and resistance towardMeloidogyne incognita was apparent. In plants infected with nematodes, tomatine in the roots was the same as in uninfected controls. Tomatine concentration, as measured by bulk analysis, does not appear to play an important role in resistance toward the nematode studied.     

A novelstreptomyces species for controlling plant-parasitic nematodes

A novel species ofSteptromyces isolated from nematode suppressive soils in Costa Rica was evaluated for efficacy in controlling plant-parasitic nematodes. This isolate, designated CR-43, was shown to inhibit reproduction ofCaenorhabditis elegans in a laboratory assay. Greenhouse trials utilizing three different methods of treatment with CR-43 gave significant reductions of tomato root galling due toMeloidogyne incognita. In a field experiment in Puerto Rico, in soil naturally infested withM. incognita, CR-43-treated pepper showed significant reductions in root galling and significant increases in yield as compared to untreated controls. In a second experiment in Puerto Rico, a significant reduction in tomato root galling and a slight reduction in root galling on pepper occurred. In this trial, yields on both tomato and pepper were higher in CR-43 treatments, but these differences were not statistically significant. In both experiments populations ofRotylenchulus reniformis were reduced by CR-43 treatment. In a field trial on strawberry in Massachusetts, CR-43-treated plants had lower numbers ofPratylenchus penetrons within roots and showed a significant decrease in black root rot disease. Studies on sterile filtrates from CR-43 cultures indicated that a major determinant of CR-43 antinematodal activity was mostly thermostable macromolecules of molecular weight higher than 6000. Culture filtrates of CR-43 exhibited antifungal activity in vitro.     

Seasonal variation of exudate ofCistus ladanifer

The production of labdanum exudate byCistus ladanifer L. is highly seasonal, reaching a maximum concentration during summer and a minimum concentration in winter. Because this exudate strongly absorbs in the wavelength range of 260–400 nm (the near-UV-visible range), it may be important biologically as an UV-visible filter. Separation of exudate components has been achieved by reverse-phase high-performance liquid chromatography (HPLC).The retention times of HPLC chromatograms and the spectral characteristics (absorption and fluorescence) of the exudate identify flavonoids as the most relevant chromophores regarding the potency of the exudate as a UV-visible filter. HPLC studies show that kaempferol-3-(O)methyl, kaempferol-3,7-di(O)methyl, and apigenin-4-(O)methyl are the most enriched flavonoids in the exudate.Other flavonoids [apigenin, apigenin-7-(O)methyl, apigenin-7,4-di(O)methyl, kaempferol-3,4-di(O)methyl and kaempferol-3,7,4-tri(O)methyl] are present in the exudate as minor components, e.g., each contributes by less than 10% to total flavonoids.The ratio of kaempferols to apigenins of the exudate also shows seasonal variation (maximum value in summer and minimum in spring). However, due to the similar absorption spectra of both groups of flavonoids, this has a minor influence on the exudate's ability to filter near-UV-visible radiation.     

Analysis of trichome exudate from mite-resistant geraniums

Trichome exudate from mite-resistant geraniums (Pelargonium horlorum) was analyzed, principally by mass spectrometry and NMR spectroscopy. The exudate was found to consist of two anacardic acid derivatives,o-pentadecenylsalicylic acid ando-heptadecenylsalicylic acid. Bioassays established a moderate toxicity of these compounds to the two-spotted spider mite,Tetranychus urticae. The production of these compounds in geraniums was correlated with the two complementary dominant genes previously reported for host resistance to spider mites.     

Characterization and partial purification of attractants for nematodeOrrina phyllobia from foliage ofSolanum elaeagnifolium

An unknown attractant for the nematodeOrrina phyllobia was extracted with water from foliage ofSolanum elaeagnifolium. Stability, solubility, ionic character, and Chromatographie purification were investigated using a bioassay based on nematode aggregation in agar. Activity was nonvolatile, dialyzable, heat stable below 60 °C, and partially lost within 30 min at 100 °C. Activity was unchanged from pH 5 to 12, but was entirely lost at pH 2. Loss of activity at low pH did not appear to result from direct effects of pH on nematode behavior and was partially recovered by readjustment to pH 7. The attractive factor was most soluble in water and appeared to be cationically but not anionically exchangeable. Activity appeared to Chromatograph as several compounds by high-performance liquid chromatography employing reverse phase C₁₈ and amine-bonded columns. Various known compounds that are common toSolanum spp. or that attract other nematodes were unattractive. Extraction ofS. elaeagnifolium foliage specifically for solanaceous glycoalkaloids using methods developed forS. tuberosum did not yield an attractive product.     

Repellency of oral exudate to eastern and western spruce budworm larvae (Lepidoptera: Tortricidae)

A two-choice feeding bioassay was used to investigate the intra-specific repellency of the larval oral exudate of eastern and western spruce budworms,Choristoneura fumiferana (Clem.) andC. occidentalis Free., respectively. Results of the bioassay indicated that feeding behavior on artificial diet-drop feeding stations was modified in the presence of exudate, with feeding stations treated with conspecific exudate being avoided when an untreated station was available 3 cm away. Feeding was suppressed when a single, exudate-treated station was provided, or when the treated and untreated stations were separated by only 1 cm. The repellent effect functioned both inter- and intraspecifically. When induced to produce exudate,C. occidentalis larvae were not immediately repelled by either their own or other individuals' exudate. However, 24 hr after induction, test larvae were repelled by exudate from either source. In both species, larval oral exudate probably functions to repel conspecific competitors.     

Studies onPaecilomyces marquandii from nematode suppressive chinampa soils

Two applications of isolates ofPaecilomyces marquandii from suppressive chinampa soils or P. lilacinus from Peru, fungi that parasitize nematode eggs, generally gave better control of tomato root-knot due toMeloidogyne incognita than did a single application. The effects on root galling by each of thePaecilomyces isolates varied between experiments; however, the ovicidal potential of the three isolates did not differ significantly. Proteins specific for each of the isolates were demonstrated by SDS gel electrophoresis. The results indicate thatP. marquandii is one of the natural soil organisms that contribute to nematode suppression in the chinampa agricultural soils.     

Host recognition by entomopathogenic nematodes: Behavioral response to contact with host feces

Host recognition by entomopathogenic nematodes may occur through contact with insects' excretory products, cuticle, or gut contents. We analyzed the behavioral responses of four species of entomopathogenic nematodes during contact with feces of natural or experimental hosts. Host recognition by nematodes was manifested in alterations in the frequency and/or duration of one or more search parameters including forward crawling, headwaving, body-waving, stopping, backward crawling, head-rubbing, and headthrusting.Heterorhabditis bacteriophora andSteinernema glaseri showed behavioral responses to contact with feces of their natural hosts,Spodoptera exigua (Lepidoptera) andPopillia japonica (Coleoptera), and to the experimental hosts,Acheata domesticus (Orthoptera) andBlatella germanica (Blatteria).Steinernema carpocapsae responded only toB. germanica feces, whereas5. scapterisci did not significantly respond to any of the insect species. During contact with cockroach feces, all nematodes, exceptS. scapterisci, showed avoidance behavior. We suggest that ammonia present in cockroach feces is inhibitory to nematodes. Specific host recognition by entomopathogenic nematodes may be an important mechanism to maintain host affinities.     

Isolation and structure determination of new nematicidal triglyceride fromArgemone mexicana

The petroleum ether extract ofArgemone mexicana seeds was found to possess nematicidal activity against the plant parasitic nematodeMeloidogyne incognita. The active nematicidal compound has been isolated from the crude extract by column Chromatographic techniques and purified by TLC. Chemical structure has been determined by chemical and spectro-scopic methods to be that of a triglyceride, sn-glycerol-1-eicosa-9, 12-dien-oate-2-palmitoleate-3-linoleate.     

Repellent,toxic, and food protectant effects of pithraj,Aphanamixis polystachya extracts against pulse beetle,Callosobruchus chinensis in storage

Ground leaves, bark, seeds, and four seed extracts of pithraj,Aphanamixis polystachya (family Meliaceae), a locally grown plant in Bangladesh, were evaluated for their repellency, contact toxicity, and food protectant efficacy against adult pulse beetle (Callosobruchus chinensis L.). The seed extracts showed poor repellent effects, but high contact toxicity to adults at 72 hr after application. The ground leaves, bark, and seeds provided good protection for mung beans against pulse beetles, and the seed powder greatly reduced the F1 progeny and seed damage rates.