Teratorhabditis palmarum n. sp. (Nemata: Rhabditidae): An Associate of Rhynchophorus palmarum and R. cruentatus

Teratorhabditis palmarum n. sp., an associate of the palm weevils Rhynchophorus palmarum and R. cruentatus is described. Teratorhabditis palmarum was isolated from newly emerged adults and cocoons of R. palmarum from red-ring diseased coconut palms, Cocos nucifera, in Trinidad and Ecuador, and from red-ring diseased oil palms, Elaeis guineensis, in Colombia. Teratorhabditis palmarum was also associated internally with newly emerged adults of R. cruentatus from mature transplanted cabbage palmettos, Sabal palmetto, in Florida. Dauer juveniles of T. palmarum infested the genital capsule and body cavity of newly emerged adult female and male palm weevils. Adult nematodes isolated from cocoons and dauer juveniles from newly emerged palm weevils were cuhurable on bacterial lawns on several solid media. Females of T. palmarum have a single anteriorly directed ovary; vulva at 93-96% of the body length; short, hemispherical spicate tail; three or four teeth in the metastom; cuticle with distinct transverse punctations that change abruptly at the level of the procorpus to indentations of alternating size and arrangement; and eggs with cuticular sculpturing. Males have a crenate, peloderan bursa with a 2 + 5 + 3 pattern of bursal rays (7 extend to the margin of bursa); spicules linear, completely fused at the distal tip and dorsally for 50% of the total spicule length.     

Improved Nematode Extraction from Carrot Disk Culture

Radopholus spp. were reared in carrot tissue culture via established procedures, with slight modification. Several plant tissue maceration enzymes and flotation media (salts and sucrose) were evaluated with regard to nematode toxicity and extraction efficiency. Best extraction of viable nematodes and eggs was attained when carrot tissue infested with Radopholus citrophilus or R. similis was macerated with a mixture of 0.50% driselase and 0.50% cellulysin, w/v each, with 2.5 ml of enzyme solution based for each gram of carrot tissue. Maceration slurries containing carrot tissue and nematodes were maintained in open flasks on a rotary shaker (175 rpm) at 26 C for 24 hours. Nematodes and eggs were extracted from resultant culture slurries by flotation with MgSO₄-7H₂0 (sp gr 1.1). A protocol is presented to extract large quantities of viable burrowing nematodes and their eggs from carrot disk cultures.     

Molecular profiling of nematode associates with Rhynchophorus ferrugineus in southern Italy

A survey of nematodes associated with the red palm weevil Rhynchophorus ferrugineus was conducted in southern Italy in 2015 and 2016 in order to create a species inventory and obtain data about nematode biodiversity. A total of 70 insect samples (pupae and adults) were collected from infested Phoenix canariensis, Phoenix dactylifera, and Chamaerops humilis palms in three Italian Regions: sampling took place at 11 locations in Apulia, 1 in Basilicata, and 1 in Sardinia regions. Individual insects were dissected to determine nematode presence, and different nematode species were also recovered from red palm weevil cocoons collected at the sites in Apulia. Individual nematodes were molecularly identified by sequencing the ITS, D2‐D3 expansion domains of the 28SrRNA gene and the mitochondrial COI and inferring the phylogenetic relationships. The insect‐associated nematofauna identified belonged to the families Rhabditidae, Cephalobidae, and Diplogastridae. Just two nematode species, Teratorhabditis synpapillata and Mononchoides macrospiculum, were always found in association with adult insects and cocoons taken from all sampling sites. This paper reports on the biodiversity of the nematodes associated with R. ferrugineus and on current knowledge of the specific habitat of specialized and divergent entomophilic nematodes.     

Correlation of Edaphic Factors with Plant-parasitic Nematode Population Densities in a Forage Field

Two hundred soil samples from the A_p horizon of a reed canarygrass field overlaying several different but related soils in northern Minnesota were analyzed for plant-parasitic nematodes and 22 edaphic factors. Pratylenchus penetrans was the predominant nematode taxon. Others were Aglenchus agricola, Tylenchorhynchus spp., Heterodera trifolii, Paratylenchus spp., Tylenchus maius, and Criconemella sp. Five nematode taxa, P. penetrans, A, agricola, Tylenchorhynchus spp., H. trifolii, and Paratylenchus spp., were correlated with particle size, Tylenchus maius and Criconemella sp. were correlated with effective cation exchange capacity. Nematode field spatial arrangements were related to a combination of statistically significant positive and negative soil factor effects on the nematode populations. Contour maps derived by geostatistical techniques were used to visually validate statistically significant correlations of nematode and soil data. Contour mapping to supplement traditional statistical techniques can be used to achieve a more holistic approach to studies of nematode-soil interrelationships.     

Commensal Nematodes in the Glands,Genitalia, and Brood Cells of Bees (Apoidea)

Seven species of bees from the eastern United States, representing four families in the Apoidea, were dissected and examined for nematode associates. Dufour''s glands in females of Halictus ligatus, Augochlora pura mosieri, and Augochlorella gratiosa (Halictidae) from Florida were infested with dauer juveniles of Aduncospiculum halicti (Diplogasteridae). The Dufour''s glands of Colletes thoracicus (Colletidae) females from Maryland were infested with dauer juveniles of a new species of Koerneria sp. (Diplogasteridae), and abdominal glands of females of Andrena alleghaniensis (Andrenidae) from New York were infested with dauer juveniles of another new species of Koerneria. The lateral and median oviducts, Dufour''s glands, and poison sacs in females of Anthophora abrupta (Anthophoridae) from Maryland and Alabama were infested with dauer juveniles of a new species of Bursaphelenchus sp. (Aphelenchoididae). Cross sections of the nematode-infested poison sacs of A. abrupta revealed two types of humoral host defense reactions.     

Drip Irrigation as a Delivery System for Infestation of Field Plots with Nematodes

A drip irrigation delivery system was used to infest field sites with the plant-parasitic root-knot nematodes, Meloidogyne incognita. Juvenile or egg inocula passed through the system without blockage of emitters or harm to the nematodes. Field sites so infested were available for experimentation. Delivery of approximately 5 x 10⁴ to 10⁵ juveniles or 10⁵ to 3 x 10⁵ eggs per emitter through the drip system resulted in heavy root galling of tomatoes planted next to the drip emitters. Nematodes feeding on bacteria (Acrobeloides sp.) and on fungi (Deladenus durus) also were successfully applied through the drip system. This method has potential for uniformly infesting experimental sites with plant-parasitic or entomogenous nematodes and for manipulation of nematode community structure for soil ecological studies.     

Effects and Dynamics of a Nematode Community on Maize

Relationships between nematode density and yield and between final and preplant population levels were examined in small maize plots on sandy soils in north-central Florida. Plant-parasitic nematodes present in the community included Belonolaimus longicaudatus, Criconemella sphaerocephala, Meloidogyne incognita, Paratrichodorus minor, Pratylenchus brachyurus, and a Xiphinema sp. Plant growth--including stand count, grain yield, stalk weight, and size of young plants--often was inversely correlated (P ≤ 0.05) with densities of B. longicaudatus and occasionally with P. brachyurus, but not with densities of other species or with a range of soil variables. More severe losses in grain yields from B. longicaudatus occurred in 1987 than in 1988, although mean preplant nematode densities in February were similar in both years (4.4 vs. 3.9/100 cm³ soil). Final population densities of most nematode species were linearly related (P ≤ 0.05) to densities measured at planting or earlier. These relationships were stronger (higher r²) with the ectoparasites B. longicaudatus and C. sphaerocephala than with the endoparasites M. incognita and P. brachyurus. No significant correlations were found between population densities of different nematode species.     

Parasitism of Heterodera schachtii and Meloidogyne javanica by Hirsutella rhossiliensis in Microplots over Two Growing Seasons

Numbers of cyst and root-knot nematodes and percentage parasitism by the nematophagous fungus Hirsutella rhossiliensis were quantified in microplots over 2 years. The microplots contained either sugarbeets in loam infested with Heterodera schachtii or tomatoes in sand infested with Meloidogyne javanica. The fungus was added to half of the microplots for each crop. Although H. rhossiliensis established in both microplot soils, the percentage of nematodes parasitized did not increase with nematode density and nematode numbers were not affected by the fungus. The results indicate that long-term interactions between populations of the fungus and cyst or root-knot nematodes will not result in biological control.     

Bionomics of a Phoretic Association Between Paenibacillus sp. and the Entomopathogenic Nematode Steinernema diaprepesi

Spores of an unidentified bacterium were discovered adhering to cuticles of third-stage infective juvenile (IJ) Steinernema diaprepesi endemic in a central Florida citrus orchard. The spores were cup-shaped, 5 to 6 mm in length, and contained a central endospore. Based on 16S rDNA gene sequencing, the bacterium is closely related to the insect pathogens Paenibacillus popilliae and P. lentimorbus. However, unlike the latter bacteria, the Paenibacillus sp. is non-fastidious and grew readily on several standard media. The bacterium did not attach to cuticles of several entomopathogenic or plant-parasitic nematodes tested, suggesting host specificity to S. diaprepesi. Attachment of Paenibacillus sp. to the third-stage cuticle of S. diaprepesi differed from Paenibacillus spp. associated with heterorhabditid entomopathogenic nematodes, which attach to the IJ sheath (second-stage cuticle). The inability to detect endospores within the body of S. diaprepesi indicates that the bacterial association with the nematode is phoretic. The Paenibacillus sp. showed limited virulence to Diaprepes abbreviatus, requiring inoculation of larvae with 108 spores to achieve death of the insect and reproduction of the bacterium. The effect of the bacterium on the nematode population biology was studied in 25-cm-long vertical sand columns. A single D. abbreviatus larva was confined below 15-cm depth, and the soil surface was inoculated with either spore-free or spore-encumbered IJ nematodes. After 7 days, the proportion of IJ below 5-cm depth was seven-fold greater for spore-free IJ than for spore-encumbered nematodes. Mortality of D. abbreviatus larvae was 72% greater (P <= 0.01) for spore-free compared to spore-encumbered S. diaprepesi. More than 5 times as many progeny IJs (P <= 0.01) were produced by spore-free compared to spore-encumbered nematodes. These data suggest that the bacterium is a component of the D. abbreviatus food web with some potential to regulate a natural enemy of the insect.     

Suppression of Root-knot Nematode Populations with Selected Rapeseed Cultivars as Green Manure

Meloidogyne chitwoodi races 1 and 2 and M. hapla reproduced on 12 cultivars of Brassica napus and two cultivars of B. campestris. The mean reproductive factors (Rf), Rf = Pf at 55 days ÷ 5,000, for the three nematodes were 8.3, 2.2, and 14.3, respectively. All three nematodes reproduced more efficiently (P < 0.05) on B. campestris than on B. napus. Amending M. chitwoodi-infested soil in plastic bags with chopped shoots of Jupiter rapeseed reduced the nematode population more (P < 0.05) than amendment with wheat shoots. Incorporating Jupiter shoots to soil heavily infested with M. chitwoodi in microplots reduced the nematode population more (P < 0.05) than fallow or corn shoot treatments. The greatest reduction in nematode population density was attained by cropping rapeseed for 2 months and incorporating it into the soil as a green manure.     

Current Research on the Major Nematode Problems in Japan

Among important nematode species occurring in Japan, current research achievements with the following four nematodes are reviewed: 1) Soybean cyst nematode (SCN), Heterodera glycines - breeding for resistance, race determination, association with Cephalosporium gregatum in azuki bean disease, and isolation of hatching stimulant. 2) Potato-cyst nematode (PCN), Globodera rostochiensis - pathotype determination (Ro 1), breeding for resistance, and control recommendations. 3) Pinewood nematode (PWN), Bursaphelenchus xylophilus - primary pathogen in pine wilt disease, life cycle exhibiting a typical symbiosis with Japanese pine sawyer, Monochamus alternatus, and project for control. 4) Rice root nematodes (RRN), Hirschmanniella imamuri and H. oryzae - distribution of species, population levels in roots, and role of these nematodes in rice culture.     

In Vitro Culture and Feeding Behavior of Belonolaimus longicaudatus on Excised Zea mays Roots

A greenhouse population of the sting nematode, Belonolaimus longicaudatus, obtained from an infested golf course in California''s Coachella Valley, was surface-decontaminated and cuhured on excised roots of Zea mays supported by Gamborg''s B5 medium. At 26-27 °C the females laid eggs, and newly emerged juveniles of the second generation completed three molts within 29 days after egg deposition. Sixty days after inoculation with 60 females and 40 males, an average of 529 nematodes and 83 eggs were recovered from the culture. The feeding process consisted of probing, stylet penetration, ingestion, and stylet retraction. Feeding seemed to be necessary before egg deposition or molting occurred. The sting nematode was observed feeding exclusively as an ectoparasite and preferably at the region of cell division and elongation. Vigorous feeding by many nematodes usually caused discoloration of root tips and termination of growth.     

Host Suitability of Soybean Cultivars and Breeding Lines to Reniform Nematode in Tests Conducted in 2001

Reproduction of reniform nematode Rotylenchulus reniformis on 139 soybean lines was evaluated in a greenhouse in the summer of 2001. Cultivars and lines (119 total) were new in the Arkansas and Mississippi Soybean Testing Programs, and an additional 20 were submitted by C. Overstreet, Louisiana State Extension Nematologist. A second test of 32 breeding lines and 2 cultivars from the Clemson University soybean breeding program was performed at the same time under the same conditions. Controls were the resistant cultivars Forrest and Hartwig, susceptible Braxton, and fallow infested soil. Five treatment replications were planted in sandy loam soil infested with 1,744 eggs and vermiform reniform nematodes, grown for 10 weeks in 10 cm-diam.- pots. Total reniform nematodes extracted from soil and roots was determined, and a reproductive factor (final population (Pf)/ initial inoculum level (Pi)) was calculated for each genotype. Reproduction on each genotype was compared to the reproduction on the resistant cultivar Forrest (RF), and the log ratio [log₁₀(RF + 1) is reported. Cultivars with reproduction not significantly different from Forrest (log ratio) were not suitable hosts, whereas those with greater reproductive indices were considered suitable hosts. These data will be useful in the selection of soybean cultivars to use in rotation with cotton or other susceptible crops to help control the reniform nematode and to select useful breeding lines as parent material for future development of reniform nematode resistant cultivars and lines.     

Morphology and Description of Bursaphelenchus platzeri n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Nitidulid Beetles

Bursaphelenchus platzeri n. sp., an associate of nitidulid beetles in southern California, is described and illustrated. Adult males and females of B. platzeri n. sp. were examined by scanning electron microscopy for ultrastructural comparisons with other members of the genus. Bursaphelenchus cocophilus (red ring nematode) appears to be the closest related taxon to B. platzeri n. sp. based upon shared morphological features of the fused spicules, female tail shape, phoresy with non-scolytid beetles, and molecular analysis of the near full-length small subunit (SSU) rDNA. Unfortunately, sequence data from the D2D3 expansion segments of the large subunit (LSU) rDNA and partial mitochondrial DNA COI did not help resolve the relationship of nearest relative. In addition to significant molecular sequence differences in SSU, LSU, and COI, B. platzeri n. sp., which is an obligate fungal feeder, can be differentiated from B. cocophilus because it is an obligate parasite of palms. Bursaphelenchus platzeri n. sp. can be differentiated from all other species of Bursaphelenchus by the length and shape of the female tail and spicule morphology. The spicules are fused along the ventral midline and possess unfused cucullae; the fused unit appears to function as a conduit for sperm. Population growth of B. platzeri n. sp. was measured in a time-course experiment at 25°C in the laboratory on cultures of the fungus Monilinia fructicola grown on 5% glycerol-supplemented potato dextrose agar (GPDA). Nematode population densities rapidly increased from 25 to approximately 200,000/culture within 14 d and then plateaued for up to 28 d.     

The Effect of Endophytic Fungi on Nematode Populations in Summer-dormant and Summer-active Tall Fescue

Summer-active (continental) and summer-dormant (Mediterranean) tall fescue morphotypes are each adapted to different environmental conditions. Endophyte presence provides plant parasitic nematode resistance, but not with all endophyte strains and cultivar combinations. This study sought to compare effects of four nematode genera on continental and Mediterranean cultivars infected with common toxic or novel endophyte strains. A 6-mon greenhouse study was conducted with continental cultivars, Kentucky 31 (common toxic) and Texoma MaxQ II (novel endophyte) and the Mediterranean cultivar Flecha MaxQ (novel endophyte). Endophyte-free plants of each cultivar were controls. Each cultivar × endophyte combination was randomly assigned to a control, low or high inoculation rate of a mixed nematode culture containing stunt nematodes (Tylenchorhynchus spp.), ring nematodes (Criconemella spp.), spiral nematodes (Helicotylenchus spp.), and lesion nematodes (Pratylenchus spp.). Endophyte infection had no effect on nematode population densities. The cultivar × endophyte interaction was significant. Population densities of stunt nematode, spiral nematode, and ring nematodes were higher for Flecha MaxQ than other cultivar × endophyte combinations. Novel endophyte infection enhances suitability of Flecha MaxQ as a nematode host.     

Survival and Infectivity of Bursaphelenchus xylophilus in Wood Chip-Soil Mixtures

To determine the effect of soil environment on the life stages and total numbers of Bursaphelenchus xylophilus, nematode-infested wood chips alone and mixed with soil were incubated at 12 and 20 C. Nematodes were extracted at 2-week intervals for 12 weeks. Numbers of nematodes and percentage of third-stage dispersal larvae were greater at 12 C and in chips without soil. Percentage of juveniles of the propagative cycle was greater at 20 C and in chips with soil. Although B. xylophilus survived in chips with soil for 12 weeks, nematode numbers and life stage percentages changed little over time. To determine if B. xylophilus was capable of infecting wounded roots, infested and uninfested chips were mixed with soil in pots with white and Scots pine seedlings. Trees were maintained at 20 and 30 C and harvested at mortality or after 12 weeks. Only seedlings treated with infested chips contained nematodes. In field experiments, planted seedlings were mulched with infested chips to determine if nematodes would invade basal stem wounds. Among these trees, Scots pine was more susceptible than white or red pines to infection and mortality.     

Cereal Cyst Nematode (Heterodera avenae) on Oats. II. Early Root Development and Nematode Tolerance

The effect of Heterodera avenae infestation on early seminal and lateral root growth was examined in four oat genotypes differing in tolerance to H. avenae. Recently emerged seminal roots were inoculated with a range of H. avenae larval densities, then transferred a hydroponic system to remove the effect of later nematode penetration on root development. Intolerance to H. avenae was assessed in terms of impairment of seminal root extension resulting in fewer primary lateral roots emerging from the seminal root below the zone of juvenile penetration. Tolerant plants infested with H. avenae had longer lateral root systems than infested intolerant plants. The decline in lateral root growth below the penetration zone was partly offset by increased growth above. This did not contribute to tolerance, however, as there were no differences between cultivars for this feature. Nematodes induced earlier nodal root emergence in all cultivars. Nodal root development was most advanced on the most tolerant cultivar.     

Molecular Transfer of Nematode Resistance Genes

Recombinant DNA techniques have been used to introduce agronomically valuable traits, including resistance to viruses, herbicides, and insects, into crop plants. Introduction of these genes into plants frequently involves Agrobacterium-mediated gene transfer. The potential exists for applying this technology to nematode control by introducing genes conferring resistance to nematodes. Transferred genes could include those encoding products detrimental to nematode development or reproduction as well as cloned host resistance genes. Host genes that confer resistance to cyst or root-knot nematode species have been identified in many plants. The best characterized is Mi, a gene that confers resistance to root-knot nematodes in tomato. A map-based cloning approach is being used to isolate the gene. For development of a detailed map of the region of the genome surrounding Mi, DNA markers genetically linked to Mi have been identified and analyzed in tomato lines that have undergone a recombination event near Mi. The molecular map will be used to identify DNA corresponding to Mi. We estimate that a clone of Mi will be obtained in 2-5 years. An exciting prospect is that introduction of this gene will confer resistance in plant species without currently available sources of resistance.     

Effects and Dynamics of a Nematode Community on Soybean

The relationships between densities of all members of a plant-parasitic nematode community and yield of ''Davis'' soybean and between final and preplant population levels were examined in small plots on sandy soils in north-central Florida. Plant-parasitic nematodes present in the community included Belonolaimus longicaudatus, Criconemella sphaerocephala, Meloidogyne incognita, Paratrichodorus minor, Pratylenchus brachyurus, and Xiphinema sp. Plant growth, including stand count, soybean yield (kg/ha), and size of young plants, was occasionally inversely correlated (P ≤ 0.05) with densities of B. longicaudatus or P. brachyurus, but not with densities of other species or with a range of soil variables. The nature of this relationship varied with season, with more severe stand losses noted during 1987 than in 1988. Final population densities (Pf) of most nematode species showed significant (P ≤ 0.05) linear relationships to densities measured at planting or earlier (Pi). These relationships were stronger (higher r²) with the ectoparasite B. longicaudatus than with the endoparasites M. incognita and P. brachyurus. Criconemella sphaerocephala declined under soybean cultivation, reaching levels near zero after two seasons. A quadratic model showed an improvement (P ≤ 0.05) over the linear model in describing the relationship between Pf and Pi measured at planting for B. longicaudatus, and gave a better indication of the leveling off of Pf at high values of Pi.     

Descriptions of Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina) from Haryana,India

Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and spermathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b’ = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c’ = 2.0 to 2.9, stylet = 11 to 12 µm, excretory pore in the region of median pharyngeal bulb, 43 to 47 µm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b’ = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c’ = 2.7 to 3.5, stylet = 6 to 7 µm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.