Carbon Dioxide and Temperature Gradielits in Baermann Funnel Extraction of Rotylenchulus reniformis

Vermiform Rotylenchulus reniformis were anesthetized in water by 10-40% CO₂ but were fully motile for 24 hours in water below 5% CO₂. When air containing 2.5% CO₂ was blown onto agar, nematodes accumulated at the point of highest CO₂ concentration. Nematodes also accumulated when chilling (0.2-1 C) of agar by the gas flow at the accumulation point was offset with heat from a fiber optic. In Baermann funnels containing R. reniformis in silt loam and sandy clay loam soils, CO₂ in funnel water increased during 24 hours from 0 to ca. 1%; more CO₂ accumulated below the soil layer than above. Bubbling air with 2.5% CO₂ into water below soil in covered funnels increased the CO₂ gradient and increased nematode extraction, whereas bubbling air without CO₂ below soil purged CO₂ from the water and decreased nematode extraction. Manipulation of CO₂ within funnels usually increased extraction by only 30% and never by more than 3-fold. Controlling temperature gradients consistently increased extraction by 2-30-fold.     

The Influence of Environmental Factors on the Respiration of Plant-Parasitic Nematodes

Respiration of selected nematode species was measured relative to CO₂ level, temperature, osmotic pressure, humidity, glucose utilization and high ionic concentrations of sodium and potassium.In general, respiration was stimulated most by the dominant environmental factors at levels near those expected in the nematode''s "natural" habitat. Soil-inhabiting nematodes utilized O₂, most rapidly with high (1-2%) CO₂ whereas a foliar nematode (Aphelenchoides ritzemabosi) did so with 0.03% CO₂, the concentration typically found in air. Temperature optima for respiration corresponded closely to those for other activities. Ditylenchus dipsaci and Pratylenchus penetrans adults and Anguina tritici and A. agrostis second-stage larvae respired within the range of osmotic pressures from 0 to 44.8 arm and respiration of their drought-resistant stages was stimulated by increasing osmotic pressure which accompanies the onset of drought. Rehydration of A. tritici and A. agrostis larvae with RH as low as 5% stimulated measurable respiration. Glucose utilization from liquid medium by A. tritici larvae or A. ritzembosi was not detectable. Supplemental Na⁺ stimulated respiration of Anguina tritici, K⁺ did not.     

The Effect of Single and Combined Heat and CO2 Stimuli at Different Ambient Temperatures on the Behavior of Two Plant-Parasitic Nematodes

Pratylenchus penetrans and Ditylenchus dipsaci were reared at 15-16 C, and their behavior towards single and combined heat and CO₂ stimuli was studied at ambient temperatures of 8.6 and 27.3 C. At the lower temperature, attractivity of the heat source was prevalent in both species, but CO₂ was also attractive. At the higher ambient temperature (27.3 C), the reaction to CO₂ was more positive and more rapid than to heat. In fact, at this temperature only D. dipsaci was attracted to the heat source, whereas P. penetrans did not react positively. The combined stimulation of heat and CO₂ caused D. dipsaci to aggregate more strongly than did a single stimulus; this applied to both ambient temperatures. For P. penetrans exposed to the low temperature (8.6 C), the combined stimuli were about as attractive as was the better of the single stimuli; i.e., heat. At the high temperature (27.3 C), the combined stimulation was less effective than the better of the single stimuli; i.e., CO₂. At this ambient temperature, the thermonegative reaction seems to dominate over the CO₂-positive one. The reaction of D. dipsaci was generally stronger in all experimental variants than that of P. penetrans. Insofar as temperature gradients play a role in locating host plant roots, their efficacy would seem to be restricted to a favorable temperature range. Within this range, combined heat and CO₂ stimuli might improve attractivity.     

Pratylenchus penetrans (Cobb) Populations as Influenced by Microorganisms and Soil Amendments

Numbers of Pratylenchus penetrans in sterilized soil decreased significantly 2 weeks after the addition of 1% w/w (700 ppm N) nonsterile soybean meal (SBM), or sterilized SBM in combination with selected microorganisms. Sterilized SBM had no effect on nematode populations in steamed soil. Bacteria and fungi in the presence of SBM were more effective than the actinomycetes tested, causing up to 96-100% reduction in nematode populations. Simpler nitrogenous compounds included KNO₂, Ca(NO₃)₂, NH₄NO₃, (NH₄)₂CO₃, urea, and peptone, decreased nematode populations with variable effectiveness when added to steamed soil at 700 ppm N; KNO₂ was the most nematicidal.     

Optimal Release Rates for Attracting Meloidogyne incognita,Rotylenchulus reniformis,and Other Nematodes to Carbon Dioxide in Sand

Movement of vermiform stages of Meloidogyne incognita, Rotylenchulus reniformis, Ditylenchus phyllobius, Steinernema glaseri, and Caenorhabditis elegans in response to carbon dioxide was studied in 40- and 72-mm-long cylinders of moist sand inside 38-mm-d acrylic tubes. Meloidogyne incognita, R. reniformis, and S. glaseri were attracted to CO₂ when placed on a linear gradient of 0.2%/cm at a mean CO₂ concentration of 1.2%. When CO₂ was delivered into the sand through a syringe needle at flow rates between 2 and 130 μl/minute, the optimal flow rate for attracting M. incognita and R. reniformis was 15 μl/minute, and maximal attraction of the two species from a distance of 52 mm was achieved after 29 and 40 hours, respectively. After 24 hours, a total CO₂ volume of 20 cm³ was sufficient to induce 96% of all M. incognita introduced to move into the half of the cylinder into which CO₂ was delivered and more than 75 % to accumulate in the 9 cm³ of sand volume nearest the source. Results indicate it may be possible to use a chemical or biological source of CO₂ to attract nematodes to nematicide granules or biocontrol agents.     

Meloidogyne hapla in Organic Soil: Effects of Environment on Hatch,Movement and Root Invasion

Using new techniques, hatch and movement of Meloidogyne hapla and nematode invasion o f lettuce roots growing in organic soil were studied under controlled soil conditions of temperature, moisture, O₂ and CO₂. When O₂ levels of 2.7, 5, 10, 21 and 40% with CO₂ maintained at 0.03% were used, O₂ below 21% or at 40% reduced nematode activities compared with those at 21%. When CO₂ levels of 0.03, 0.33, 2.8, 10 and 30% with O₂ maintained at 21% were used, all levels above 0.03% CO₂ resulted in less activity than at 0.03% except for more invasion at 0.33% than at 0.03%. Results suggested M. hapla was tolerant of CO₂ below 10% but adversely affected by 30% CO₂. Effect of O₂ was influenced by the level of CO₂ present. No larvae invaded roots at 3.2% O₂ and 18.6% CO₂ but hatch and movement occurred. Night and day temperatures of 21.1 and 26.7 C were more favorable for movement and invasion than 15.5 and 21.1 C, 26.7 and 32.2 C or 26.7 and 32.2 C. Optimum moisture for movement was 80 cm suction and for invasion was 100 cm.     

Decomposition of Plant Debris by the Nematophagous Fungus ARF

In the study of the biological control of plant-parasitic nematodes, knowledge of the saprophytic ability of a nematophagous fungus is necessary to understand its establishment and survival in the soil. The objectives of this study were (i) to determine if the nematophagous fungus ARF (Arkansas Fungus) shows differential use of plant residues; and (ii) to determine if ARF still existed in the soil of a field in which ARF was found originally and in which the population level of Heterodera glycines had remained very low, despite 15 years of continuous, susceptible soybean. Laboratory studies of the decomposition of wheat straw or soybean root by ARF were conducted in two separate experiments, using a CO₂ collection apparatus, where CO₂-free air was passed through sterilized cotton to remove the microorganisms in the air and then was passed over the samples, and evolved CO₂ was trapped by KOH. Milligrams of C as CO₂ was used to calculate the percentage decomposition of the plant debris by ARF. Data indicated ARF decomposed 11.7% of total organic carbon of the wheat straw and 20.1% of the soybean roots in 6 weeks. In the field soil study, 21 soil samples were taken randomly from the field. Only 3 months after the infestation of the soil with H. glycines, the percentage of parasitized eggs of H. glycines reached 64 ± 19%, and ARF was isolated from most parasitized eggs of H. glycines. Research results indicated ARF could use plant residues to survive.     

The Role of Microbes Associated with Chicken Litter in the Suppression of Meloidogyne arenaria

The role of microbes associated with chicken litter in the suppression of Meloidogyne arenaria in amended soil was investigated. Amended soil treatments were prepared, including combinations of sterile and nonsterile chicken litter and soil. Microbial biomass in different treatments was compared by measuring carbon dioxide evolution. There was less CO₂ evolved in sterile litter than in nonsterile litter treatments. Tomato seedlings cv. Rutgers were transplanted into soil mixtures and inoculated with 2,000 M. arenaria eggs. After 10 days, fewer second-stage juveniles (J2) had penetrated the roots in soils amended with nonsterile litter than sterile litter. The effects of sterile and nonsterile litter-amended soil solutions on M. arenaria eggs and J2 were observed over a period of 6 days. A lower percentage of eggs remained apparently healthy in nonsterile than in sterile-amended soil solutions over 6 days. Microbial degradation of the egg shells was apparent. Fewer J2 survived in sterile- and nonsterile-amended-soil solutions as compared to water controls.     

Evaluation of Dry Ice as a Potential Cryonematicide for Meloidogyne incognita in Soil

Solid CO₂ (dry ice) was added to pots containing soil that was infested either with eggs of the root-knot nematode, Meloidogyne incognita, or with tomato (Lycopersicon esculentum ''Rutgers'') root fragments that were infected with various stages of the nematode. Two hours after dry ice was added, thermocouples in the soil recorded temperatures ranging from -15 °C to -59 °C. One day after treatment with the dry ice, the temperature of the soil was allowed to equilibrate with that of the greenhouse, and susceptible tomato seedlings were planted in pots containing infested soil treated or untreated (controls) with dry ice. After 5 weeks, roots were removed from the pots and nematode eggs were extracted and counted. Plants grown in soil infested with eggs and receiving dry ice treatment had less than 1% of the eggs found in the controls; plants from soil infested with root fragments and receiving dry ice treatment had less than 4% of the eggs found in controls. Dry ice used to lower soil temperature may have potential as a cryonematicide.     

Gaseous Requirements for Postparasitic Development of Romanomermis culicivorax

The development of postparasitic stages of Romanomermis culicivorax was studied under various concentrations of oxygen and carhon dioxide. The nematode developed poorly if only nitrogen was supplied; only one-third molted and all died eventually. In the presence of 5% CO₂ - 95% N₂, development was normal; most nematodes molted and oviposited with respective mean developmental times of 32 and 50 d. Addition of 0.2% O₂ stimulated development; molting and oviposition commenced at days 18 and 41, respectively. There was an additional stimulation of development by increasing amounts of O₂ up to 1%, but concentrations greater than 1% produced no additional stimulation. Carbon dioxide was required for development after exsheathment under anaerobic conditions or O₂ concentrations less than 1%. Oxygen or CO₂ were not required for embryological development or egg hatch. It is suggested that post-parasitic stages function as facultative anaerobes,     

Carbon Partitioning in Soybean Infected with Meloidogyne incognita and M. javanica

Seven-day-old seedlings of two cultivars (Cristalina and UFV ITM1) of Glycine max were inoculated with 0, 3,000, 9,000, or 27,000 eggs of Meloidogyne incognita race 3 or M. javanica and maintained in a greenhouse. Thirty days later, plants were exposed to ¹⁴CO₂ for 4 hours. Twenty hours after ¹⁴CO₂ exposure, the root fresh weight, leaf dry weight, nematode eggs per gram of root, total and specific radioactivity of carbohydrates in roots, and root carbohydrate content were evaluated. Meloidogyne javanica produced more eggs than M. incognita on both varieties. A general increase in root weight and a decrease in leaf weight with increased inoculum levels were observed. Gall tissue appeared to account for most of the root mass increase in seedlings infected with M. javanica. For both nematodes there was an increase of total radioactivity in the root system with increased levels of nematodes, and this was positively related to the number of eggs per gram fresh weight and to the root fresh weight, but negatively related to leaf dry weight. In most cases, specific radioactivities of sucrose and reducing sugars were also increased with increased inoculum levels. Highest specific radioactivities were observed with reducing sugars. Although significant changes were not observed in endogenous levels of carbohydrates, sucrose content was higher than reducing sugars. The data show that nematodes are strong metabolic sinks and significantly change the carbon distribution pattern in infected soybean plants. Carbon partitioning in plants infected with nematodes may vary with the nematode genotype.     

Impact of Meloidogyne incognita on Physiological Efficiency of Vitis vinifera

Four-week-old French Colombard plants rooted from green cuttings were inoculated with 0, 1,000, 2,000, 4,000, or 8,000 Meloidogyne incognita second-stage juveniles and maintained at 25 C night and 30 C day. Leaf area and dry weight and the rates of photosynthesis, stomatal conductance, and internal leaf CO₂ concentration were measured at intervals up to 59 days after inoculation. Nematode stress dosage, measured as the product of cumulative number of juveniles and females and their total energy (calories) demand, was up to 3.4 kcal and accounted for up to 15% of the energy assimilated by the plants. There was a decline in the rate of leaf area expansion and leaf, stem, shoot, root (excluding nematode weight), and total plant dry weight with increasing nematode stress. Root weight including nematodes was not affected. Total respiration, plant photosynthesis, energy assimilated into plant tissue and respiration, and gross production efficiency decreased significantly with nematode stress. Photosynthetic rate, transpiration rate, stomatal conductance, and internal CO₂ concentration were not affected. This study demonstrates that the energy demand for growth and reproduction of M. incognita accounts for a significant portion of the total energy entering the plant system. As a result, less energy is partitioned into leaf area expansion which, in turn, affects the energy entering the system and results in decreased productivity of nematode-infected grape vines.     

Respiration Rate of Steinernema feltiae Infective Juveniles at Several Constant Temperatures

Respiration was measured in dauer stages of the insect-parasitic nematode Steinernema feltiae (= Neoaplectana carpocapsae) at 7, 17, and 27 C. Respiration, Q₁₀, and nematode viability were temperature dependent. Mean O₂ consumption for 5 × 10⁵ nematodes the first 24 hours was 0.27 ml at 7 C, 0.83 ml at 17 C, and 2.68 ml at 27 C. The Q₁₀ was 3.10 for 7-17 C and 3.24 for 17-27 C. Some nematodes died during 2, 14, and 21 days at 27, 17, and 7 C, respectively. The respiratory quotient was below 1 at all temperatures tested. A standard asymptotic model is expressed as oxygen consumed = 2.77 * {1 - exponent[-time * exponent(-B + C * temperature)]}; where 2.77 is the maximum response at 27 C. This model estimates nematode O₂ consumption and viability at storage temperatures between 7 and 27 C. The nematodes died when the O₂ concentration reached 0.5 ml/5 × 10⁵ nematodes. This model may be used to predict O₂ requirements of S. feltiae infective juveniles when stored as a waterless concentrate.     

Leaching Soluble Salts Increases Population Densities of Tylenchulus semipenetrans

The effect of salinity on population densities of Tylenchulus semipenetrans was measured on 3-month-old salt-tolerant Rangpur lime growing on either loamy sand, sand, or organic mix and on 4-month-old salt-sensitive Sweet lime in organic mix. Salinity treatments were initiated by watering daily with 25 mol/m³ NaCl + 3.3 mol/m³ CaCl₂ for 3 days and every other day with 50 mol/m³ NaC1 + 6.6 mol/m³ CaC1₂ for one week, with no salt (NS) treatments as controls. Salinity was discontinued in one treatment (DS) by leaching with tap water prior to inoculation with nematodes, whereas the continuous salinity (CS) treatment remained unchanged. Overall, in Rangpur lime organic soil supported the highest population densities of T. semipenetrans, followed by loamy sand and sand. The DS treatment resulted in the highest (P ≤ 0.05) mean population densities of T. semipenetrans in the three soil types. Similarly, the DS treatment in Sweet lime resulted in the highest (P ≤ 0.05) nematode populations. The DS treatment predisposed citrus to nematode infection through accumulated salt stress, whereas leaching soluble salt in soil solution offered nematodes a suitable nonosmotic habitat. Nematode females under the DS treatment also had the highest (P ≤ 0.05) fecundity.     

Effects of Nematophagous Fungi on Numbers and Death Rates of Bacterivorous Nematodes in Arable Soil

In a series of microcosm experiments with an arable, sandy loam soil amended with sugarbeet leaf, the short-term (8 weeks) dynamics of numbers of nematodes were measured in untreated soil and in γ-irradiated soil inoculated with either a field population of soil microorganisms and nematodes or a mixed population of laboratory-propagated bacterivorous nematode species. Sugarbeet leaf stimulated an increase in bacterivorous Rhabditidae, Cephalobidae, and a lab-cultivated Panagrolaimus sp. Differences were observed between the growth rates of the nematode population in untreated and γ-irradiated soils, which were caused by two nematophagous fungi, Arthrobotrys oligospora and Dactylaria sp. These fungi lowered the increase in nematode numbers due to the organic enrichment in the untreated soil. We estimated the annually produced bacterivous nematodes to consume 50 kg carbon and 10 kg nitrogen per ha, per year, in the upper, plowed 25 cm of arable soil.     

Effects of Meloidogyne incognita on Nitrogen Fixation in Soybean

The effects of a North Carolina population of Meloidogyne incognita on N₂ fixation on root-knot-susceptible ''Lee 68'' and moderately resistant ''Forrest'' soybean were evaluated 50, 75, I00, and 135 days after inoculation with nematodes. Nematodes stimulated N₂ fixation in Lee 68 by 50 days and in Forrest by 75 days. At all other intervals, N₂ fixation was either depressed or unaffected by nematodes. Additional observations indicate that the susceptibility of Lee 68 is associated with greater rates of penetration by larvae and more favorable responses of host tissues to nematodes than occur in Forrest. With time, however, the histological reactions of both hosts became less favorable for nematode development. Resistant or hypersensitive responses became common in Forrest by 75 days but not in Lee 68 until 90 days after inoculation. This population of M. incognita may stimulate N₂ fixation at a specific time interval and depress it at others; therefore, disease of susceptible soybeans caused by this nematode is probably not primarily due to a net loss of fixed nitrogen but to pathogenicity similar to that which occurs on nonlegume hosts.     

Effect of Crotalaria juncea Amendment on Nematode Communities in Soil with Different Agricultural Histories

Effect of sunn hemp (Crotalaria juncea) hay amendment on nematode community structure in the soil surrounding roots of yellow squash (Cucurbita pepo) infected with root-knot nematodes was examined in two greenhouse experiments. Soils were from field plots treated long-term (LT) with yard-waste compost or no yard-waste compost in LT experiment, and from a short-term (ST) agricultural site in ST experiment. Soils collected were either amended or not amended with C. juncea hay. Nematode communities were examined 2 months after squash was inoculated with Meloidogyne incognita. Amendment increased (P < 0.05) omnivorous nematodes in both experiments but increased only bacterivorous nematodes in ST experiment (P < 0.05), where the soil had relatively low organic matter (<2%). This effect of C. juncea amendment did not occur in LT experiment, in which bacterivores were already abundant. Fungivorous nematodes were not increased by C. juncea amendment in either experiment, but predatory nematodes were increased when present. Although most nematode faunal indices, including enrichment index, structure index, and channel index, were not affected by C. juncea amendment, structure index values were affected by previous soil organic matter content. Results illustrate the importance of considering soil history (organic matter, nutrient level, free-living nematode number) in anticipating changes following amendment with C. juncea hay.     

Respiratory Physiology of a Facultative Anaerobe, Romanomermis culicivorax

The respiratory physiology of postparasitic larval and adult Romanornermis culicivorax was studied by manometric and polarographic methods. Endogenous respiration rates were relatively low and unaffected by postemergent development. The respiratory quotient (RQ) of larvae and young adults was 0.4 but increased to 0.7 about 3 wk after emergence. Exogenous glucose (0.02 mM) had no effect on QO₂ or RQ. Respiration of adults and larvae was completely inhibited by KCN (l mM) but not by NaN₃ (l mM) or 2,4-DNP (0.1 mM). The nematodes survived exposure to cyanide (l mM) for 18 h. Carbon dioxide (10%) inhibited respiration. The postparasitic stages of the nematode were mixed respiratory regulators-conformers. Exposure to anaerohic conditions resulted in an increased postanaerobic oxygen consumption which persisted for 3.5 h. The experiments confirmed that the postemergent stages of the nematode are facultative anaerobes.     

Repulsion of Meloidogyne incognita by Alginate Pellets Containing Hyphae of Monacrosporium cionopagum,M. ellipsosporum,or Hirsutella rhossiliensis

The responses of second-stage juveniles (J2) of Meloidogyne incognita race 3 to calcium alginate pellets containing hyphae of the nematophagous fungi Monacrosporiura cionopagum, M. ellipsosporum, and Hirsutella rhossiliensis were examined using cylinders (38-mm-diam., 40 or 72 mm long) of sand (94% <250-μm particle size). Sand was wetted with a synthetic soil solution (10% moisture, 0.06 bar water potential). A layer of 10 or 20 pellets was placed 4 or 20 mm from one end of the cylinder. After 3, 5, or 13 days, J2 were put on both ends, on one end, or in the center; J2 were extracted from 8-ram-thick sections 1 or 2 days later. All three fungal pellets were repellent; pellets without fungi were not. Aqueous extracts of all pellets and of sand in which fungal pellets had been incubated were repellent, but acetone extracts redissolved in water were not. Injection of CO₂ (20 μl/minute) into the pellet layer attracted J2 and increased fungal-induced mortality. In vials containing four randomly positioned pellets and 17 cm³ of sand or loamy sand, the three fungi suppressed the invasion of cabbage roots by M. javanica J2. Counts of healthy and parasitized nematodes observed in roots or extracted from soil indicated that, in the vial assay, the failure of J2 to penetrate roots resulted primarily from parasitism rather than repulsion. Data were similar whether fungal inoculum consisted of pelletized hyphae or fungal-colonized Steinernema glaseri. Thus, the results indicate that nematode attractants and repellents can have major or negligible effects on the biological control efficacy of pelletized nematophagous fungi. Factors that might influence the importance of substances released by the pellets include the strength, geometry, and duration of gradients; pellet degradation by soil microflora; the nematode species involved; and attractants released by roots.     

Influence of Glomus intraradices and Soil Phosphorus on Meloidogyne incognita Infecting Cucumis melo

The interaction among Glomus intraradices, Meloidogyne incognita, and cantaloupe was studied at three soil phosphorus (P) levels in a greenhouse. All plants grew poorly in soil not amended with P, regardless of mycorrhizal or nematode status. In soil amended with 50 μg P /g soil, M. incognita suppressed the growth of nonmycorrhizal plants by 84%. In contrast, growth of mycorrhizal plants inoculated with M. incognita was retarded by only 21%. A similar trend occurred in plants grown in soil with 100 μg P /g soil. Mycorrhizal infection had no effect on the degree of root-knot gall formation and did not affect the number of nematode eggs per egg mass. Mineral levels in plant shoots generally declined as soil P levels increased and were not significantly influenced by G. intraradices or M. incognita.