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.     

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.     

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.     

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.     

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.     

Development of the False Root-knot Nematode,Nacobbus aberrans, on Sugarbeet

The duration of the embryogenic development of Nacobbus aberrans (= N. batatiformis) took 9-10 days at 25 C and 51 days at 15 C. The J₁ molted in the egg; hence the Je emerged from the egg. The effect of distilled water attd root leachates of kochia and sugarbeet was investigated at 5, 10, 15, 20, and 25 C. Root leachates did not significantly affect the percent of cumulative hatch of eggs, but temperature did significantly affect emergence of juveniles (p = 0.05). Less than 1, 5, and 20% of eggs hatched at 5, 10, and 15 C, respectively. The percent of cumulative hatch at 20 C was four times greater than at 15 C, while the highest percentage of juveniles emerged at 25 C. The duration of postembryogenic development from J₂ inoculation until the appearance of mature females with egg masses took 38 days, and the life cycle from egg to egg was completed in 48 days at 25 C. All immature stages, young females and males were migratory endoparasites. Young females were able to leave the root swellings, where they developed from juvenile stages, and re-enter the root, where they formed a true gall and became sedentary. Thirty days after inoculation with J₂ nematodes, specimens were detected in root tissues at 10, 15, 20, 25, and 30 C, hut not at 5 C. Five days after inoculation at 23 C ( ± 2 C), juveniles had penetrated the roots and caused slight swellings of the tip and axis of sugarbeet feeder roots. Large cavities extended from the cortical parenchyma to the periphery of the stelar area, and 50 % of the central cylinder was destroyed 25 days after inoculation at 23 C. No syncytia formation were detected in the sugarbeet root swellings infected with juveniles. Syncytia were associated only with adult females; hyperplasia, abnormal proliferation of lateral roots, and asymmetry of root structure were additional anatomical changes induced by adult females. Only very smooth annules but no cuticular ornamentations were noted by SEM on the perineal area of adult females.     

Effect of Age on Body Wall Cuticle Morphology of Heterodera schachtii Schmidt females

Fine structure of the body wall cuticle of Heterodera schachtii is compared with respect to age and body region of the female. The cuticle is more complex than previously reported. In newly molted females only layers A, B, and C are present, but 4 weeks after the final molt a thin D layer is present between the midbody and base of the cone. This D layer is absent in the cone of H. schachtii, regardless of age. As females age, an additional layer E is produced and includes zones E₁ and E₂. Zone El apparently is unique to H. schachtii, whereas E₂ is likely to be homologous with a similar layer in Atalodera. In the cone of old females (ca. 8 weeks after the final molt) of H. schachtii, the two zones become irregular in shape and comprise bullae. The presence of a thin D layer in Heterodera strengthens the previous hypothesis of a single ancestor of cyst nematodes.     

Effects of Oxygen and Temperature on the Activity and. Survival of Nothanguina phyllobia

The effects of oxygen and temperature on the activity and survival of infective forth-stage juveniles of Nothanguina phyllobia Thorne were examined in aqueous suspension. Rate of movement was not affected by a wide range of O₂ concentration (0.8-8.6 ppm). Activity decreased below 0.8 ppm 0 2, and at 0.15 ppm O₂ nematodes became motionless. Activity increased as a linear function of temperature up to a thermal optimum of 24 C; beyond 24 C activity decreased. Survival was greatly prolonged at low temperature. At 23 C, 50% mortality occurred within 7 d, whereas at 4 C, 70% survived after 98 d.     

Long-Term Conditioning to Elevated pCO2 and Warming Influences the Fatty and Amino Acid Composition of the Diatom Cylindrotheca fusiformis

The unabated rise in anthropogenic CO₂ emissions is predicted to strongly influence the ocean’s environment, increasing the mean sea-surface temperature by 4°C and causing a pH decline of 0.3 units by the year 2100. These changes are likely to affect the nutritional value of marine food sources since temperature and CO₂ can influence the fatty (FA) and amino acid (AA) composition of marine primary producers. Here, essential amino (EA) and polyunsaturated fatty (PUFA) acids are of particular importance due to their nutritional value to higher trophic levels. In order to determine the interactive effects of CO₂ and temperature on the nutritional quality of a primary producer, we analyzed the relative PUFA and EA composition of the diatom Cylindrotheca fusiformis cultured under a factorial matrix of 2 temperatures (14 and 19°C) and 3 partial pressures of CO₂ (180, 380, 750 μatm) for >250 generations. Our results show a decay of ~3% and ~6% in PUFA and EA content in algae kept at a pCO₂ of 750 μatm (high) compared to the 380 μatm (intermediate) CO₂ treatments at 14°C. Cultures kept at 19°C displayed a ~3% lower PUFA content under high compared to intermediate pCO₂, while EA did not show differences between treatments. Algae grown at a pCO₂ of 180 μatm (low) had a lower PUFA and AA content in relation to those at intermediate and high CO₂ levels at 14°C, but there were no differences in EA at 19°C for any CO₂ treatment. This study is the first to report adverse effects of warming and acidification on the EA of a primary producer, and corroborates previous observations of negative effects of these stressors on PUFA. Considering that only ~20% of essential biomolecules such as PUFA (and possibly EA) are incorporated into new biomass at the next trophic level, the potential impacts of adverse effects of ocean warming and acidification at the base of the food web may be amplified towards higher trophic levels, which rely on them as source of essential biomolecules.     

Populations of Pratylenchus penetrans Relative to Decomposing Nitrogenous Soil Amendments

Populations of Pratylenchus penetrans decreased in soil following addition of 70 and 700 ppm N in the form of nitrate, nitrite, organic nitrogen, or ammonium compounds. Nitrate was less effective than other nitrogen carriers. Population reduction is principally attributed to ammonification during decomposition. This hypothesis is supported by chromatographic analyses of soil atmospheres, survival of nematodes in pure CO₂ and N₂, inverse relationship of CO₂, content in amended soils to nematode populations, and direct relationship of NH₃-N content of amended soils to nematode populations.     

Metabolism of Glycogen and Neutral Lipids by Aphelenchus avenae and Caenorhabditis sp. in Aerobic,Microaerobic and Anaerobic Environments

Starving Aphelenchus avenae survived 3-4 weeks in microaerobic and anaerobic environments, but Caenorhabditis sp. survived less than 80 hr. Aerobically, both nematodes metabolize neutral lipid reserves: there was no microaerobic ( <5% O₂) or anaerobic neutral lipid catabolism. Early in anaerobiosis both nematodes utilized endogenous glycogen. Caenorhabditis sp. depleted the glycogen and died. A. avenae under oxygen stress longer than 120 hr entered cryptobiosis, during which there was neither measurable O₂ uptake nor glycogen or neutral lipid utilization, Only when re-aerated, did A. avenae recover and resume "''normal" metabolism.     

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.     

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.     

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.     

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.     

Food Consumption of the Free-Living Aquatic Nematode Pelodera chitwoodi

A Cartesian diver respirometer was used to measure O₂ uptake and respiratory quotients at 25 C. Respiratory quotients were about 0.70 in starved nematodes, and 0.80 in third-stage and adult nematodes that had fed on bacteria. The energy output as measured by O₂ uptake was inversely related to the concentration of bacteria in the medium, indicating reduction in feeding effort. Feeding bacteria to third-stage nematodes in divers quickly resulted in peak respiration rates averaging 6.4 nl O₂/μg wet weight nematode per hour (QO₂) or six times the endogenous rate. In about 4 hr, the rates fell and then stabilized at a QO₂ of 2.5. Adult males fed bacteria in divers had a peak QO₂ of 2.8 or twice the starved rate. Adult females fed bacteria had a peak QO₂ of 3.7. Starving adult males and third-stage larvae were estimated to lose 2.4% and 1.4%, respectively, of their body weight per day in the form of fat based on the caloric equivalent o f oxygen used and a respiratory quotient of 0.70. The caloric content of the bacteria fed to nematodes in divers was determined. It was then calculated that both third-stage larvae and adult males ingested bacteria equivalent to 4.4 × 10⁻⁵ cal/μg wet weight nematode tissue per hour when feeding. Of the bacterial calories ingested, the larvae used 27% and adults 21% for respiration. It was estimated that males ingested 3.1 × 10⁶ bacteria and females 10 × 10⁶ bacteria during an 8-day life span.     

Validation of a Model for Prediction of Host Damage by Two Nematode Species

Plant roots were mechanically injured or subjected to nematode parasitism to test the model of host damage by two nematode species: y = m'' + (l - m'')c''z₁^P₁₁z₂^P₁₂ for y ≤ 1.0 and y = 1.0 for y > 1.0, where m'' = m₁ + (m₂ - m₁) (1 - y₂)/[(1 - y₁) + (l - y₂)] and c'' = (z₁^-T₁ + z₂^-T₂)/2. Damage functions for greenhouse-grown radish plants (cv. Cherry Belle) mechanically injured with small or large steel needles were used to predict growth of plants injured by both needles. Growth predictions accounted for 94%, 87%, and 82% of mean treatment variation in plant height, stem weight, and root weight, respectively. Cowpea (cv. California Blackeye No. 5) damage functions, based on preplant population levels of Meloidogyne incognita and M. javanica, were used to predict seed yield of plants concomitantly infected with various levels of each species. Single species damage functions and population growth curves indicated significant host resistance to M. incognita and significantly lower virulence of that species compared to M. javanica. Model predictions accounted for 88% of mean seed yield variation in two-species treatments. In a separate experiment, mean top weights of 30-day-old cowpea plants, nniformly inoculated with 20,000 M. javanica eggs, increased with increasing levels of concomitantly inoculated M. incognita eggs. It is speculated that competitive interactions between M. incognita and M. javanica mitigated host damage by the more virulent species.     

Physiological Response of Resistant and Susceptible Vitis vinifiera Cultivars to Meloidogyne incognita

The effect of Meloidogyne incognita on growth, general physiological response, and the concentration of reducing and nonreducing sugars at the nematode feeding sites of French Colombard (susceptible) and Thompson Seedless (moderately resistant) Vitis vinifiera cultivars was studied up to 2,100 degree-days (DD-base 10 C). Nematode stress dosage, measured as the product of cumulative number of juveniles and females and their total energy (calories) demand, accounted for up to 15 and 10% of the energy assimilated by French Colombard and Thompson Seedless plants, respectively. Total leaf area, total carbon dioxide fixed, transpiration rate, stomatal conductance, and internal leaf CO₂ concentration were not affected, but energy assimilated into plant tissue and respiration were decreased by nematode infection in both cultivars. Energy consumed by nematodes accounted for most of the difference in total energy assimilated between infected and uninfected plants on French Colombard but not on Thompson Seedless, suggesting that the resistant cultivar may be using more energy to curtail the nematode''s activity. Nematodes did not affect the concentration of reducing sugars, but the concentration of nonreducing sugars increased in French Colombard and decreased in Thompson Seedless. This indicates that there was more translocation of photosynthate to the feeding sites of the susceptible than to those of the resistant cultivar, and may explain why M. incognita causes more damage to French Colombard than to Thompson Seedless.     

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.