Liquid Culture of the Entomogenous Nematode Steinernema feltiae with Its Bacterial Symbiont

The insect-parasitic nematode, Steinernema feltiae Filipjev strain 42, was reared in liquid culture along with its bacterial symbiont, Xenorhabdus nematophilus Thomas &Poinar. First-stage juveniles developed into reproducing adults in a maintenance salts medium containing resuspended Xenorhabdus cells and the yeast Kluyveromyces marxianus (Hansen) van der Walt or cholesterol. Cultures with media depths greater than 4 mm required aeration. Nematode populations increased as bacterial density increased. An optimal culture system was obtained when the bacteria and nematodes developed in a semidefined medium containing tryptic soy, yeast extract, and cholesterol and were incubated on a rotary shaker at 25 ± 1 C. Under these conditions, up to 86% of the final population were infective juveniles.     

Mass Culture of Subanguina picridis and Its Bioherbicidal Efficacy on Acroptilon repens

A Russian knapweed (Acroptilon repens) shoot culture system, initiated from shoot tip culture, was used to generate a source of host plant tissue for the rearing of the nematode Subanguina picridis, a biocontrol agent for Russian knapweed. Young shoots growing on solid B5G medium in petri dishes developed galls on leaves, petioles, and shoot tips 7 days after release of 50 nematodes onto the surface of the medium. After 3 months of culturing, each petri dish yielded 7,000-10,000 nematodes. In vitro cultured Subanguina picridis were virulent on greenhouse-grown Russian knapweed plants. Galls were first found on seedlings 12 days after infestation; after 2 months, 90% of seedlings were galled on leaves, petioles, and shoot tips, with 1-6 galls per seedling. Three months after shoot emergence, 64% of vegetative shoots originating from root segments were also galled by the cultured nematodes. Similarly, vegetatively regenerated shoots of Russian knapweed were also susceptible to infestation by cultured nematodes.     

Bacterial farming by the fungus Morchella crassipes

The interactions between bacteria and fungi, the main actors of the soil microbiome, remain poorly studied. Here, we show that the saprotrophic and ectomycorrhizal soil fungus Morchella crassipes acts as a bacterial farmer of Pseudomonas putida, which serves as a model soil bacterium. Farming by M. crassipes consists of bacterial dispersal, bacterial rearing with fungal exudates, as well as harvesting and translocation of bacterial carbon. The different phases were confirmed experimentally using cell counting and ¹³C probing. Common criteria met by other non-human farming systems are also valid for M. crassipes farming, including habitual planting, cultivation and harvesting. Specific traits include delocalization of food production and consumption and separation of roles in the colony (source versus sink areas), which are also found in human agriculture. Our study evidences a hitherto unknown mutualistic association in which bacteria gain through dispersal and rearing, while the fungus gains through the harvesting of an additional carbon source and increased stress resistance of the mycelium. This type of interaction between fungi and bacteria may play a key role in soils.     

Axenizing and Culturing Endomigratory Plant-Parasitic Nematodes using Pluronic F127, Including its Effects,on Population Dynamics of Pratylenchus penetrans

A non-chemical technique for surface sterilizing plant-parasitic nematodes for aseptic cultures is described. The method is most applicable to nematodes with active migratory infective stages and requires only a few starting specimens. Rate of achieving a primary aseptic culture with the technique ranged from 60%-100% depending on the conditions of the specimens collected for culturing. Aseptic cultures of species of Meloidogyne, Rotylenchuluz, Pratylenchus, and Radopholus initiated with the method remained contamination-free after 12 months of maintenance in tomato root explant or alfalfa callus cultures. Further studies of Pluronic F127, a polyol gel medium employed in the technique to confine the spread of contaminating bacteria or fungi associated with the nematodes, showed that the polyol gel was a suitable support medium for culturing corn root explant, alfalfa callus tissues, and consequently Pratylenchus species including P. agilis, P. brachyurus, P. scribneri, and P. penetrans. During the course of 10 months, P. penetrans reared in polyol-base medium followed a standard biological growth curve, multiplied to a higher population density, maintained a similar female-to-male ratio, and possessed a similar tendency to reside inside or outside host tissues as did P. penetrans reared in agar-base medium. The percentages of P. penetrans juveniles in the sub-populations residing outside or inside the host tissues reared in polyol-base medium also were similar to and fluctuated temporally in like manner as those reared in agar-base medium. Members of these sub-populations from the polyol- or agar-base were equally infective and reproductive after 9 months of culturing.     

Teratological Development in the Cephalic Anatomy of the Nematode Romanomermis culicivorax

Adults of Romanomermis culicivorax obtained from mass cultures were examined by scanning and transmission electron microscopy to determine the organization of their anterior sense organs. The normal pattern apparently consists of two lateral amphids plus six cephalic papillae. Lateral cephalic papillae contain two sense organs, each with a cuticular pore, while subdorsal and subventral papillae have three sense organs, each with a cuticular pore. About 30% of females and 80% of males examined showed aberrant developments in these sense organs. Both cuticle and underlying cells (hypodermis and neurons) are affected; some sense organs are absent while others are incompletely formed. Few aberrant worms were found in a smaller collection of worms reared in lower population densities. Perhaps aberrant forms are examples of teratological development resulting from, or promoted by, conditions used for mass rearing of biological control agents.     

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.     

Potential of Tissue Culture for Breeding Root-Knot Nematode Resistance into Vegetables

Plant protoplast technology is being investigated as a means of transferring root-knot nematode resistance factors from Solanum sisymbriifolium into the susceptible S. melongena. Solanum sisymbriifolium plants regenerated from callus lost resistance to Meloidogyne javanica but retained resistance to M. incognita. Tomato plants cloned from leaf discs of the root-knot nematode resistant ''Patriot'' were completely susceptible to M. incognita, while sections of stems and leaves rooted in sand in the absence of growth hormones retained resistance. Changes in resistance persisted for three generations. It is postulated that the exogenous hormonal constituents of the culture medium are modifying the expression of genetic resistance.     

Growth and Virulence of Steinernema glaseri Influenced by Different Subspecies of Xenorhabdus nematophilus

Three Xenorhabdus nematophilus subspecies influenced Steinernema glaseri growth profiles and growth rates, but this was not necessarily because of different bacterial growth rates. Virulence of dauer nematodes in larval Galleria mellonella varied with the number of dauers retaining bacteria and the bacterial subspecies. Virulence was least for dauers grown on X. nematophilus subsp. bovienii because of the lack of retained bacteria. Virulence was subsequently restored by culturing these nematodes on X. nematophilus subsp. poinari.     

New records of Mansonella ozzardi: a parasite that is spreading from the state of Amazonas to previously uninfected areas of the state of Acre in the Purus River region

Mansonella ozzardi infections are common in the riverside communities along the Solimões, Negro and Purus Rivers in the state of Amazonas (AM). However, little is known about the presence of this parasite in communities located in regions bordering AM and the state of Acre. The prevalence rate of M. ozzardi infections was determined in blood samples from volunteers according to the Knott method. A total of 355 volunteers from six riverine communities were enrolled in the study and 65 (18.3%) were found to be infected with M. ozzardi. As expected, most of the infections (25%) occurred in individuals involved in agriculture, cattle rearing and fishing and an age/sex group analysis revealed that the prevalence increased beginning in the 40-50-years-of-age group and reached 33% in both sexes in individuals over 50 years of age. Based on the described symptomatology, articular pain and headache were found to be significantly higher among infected individuals (56 and 65% prevalence, respectively, p < 0.05). Sera from volunteers were subjected to ELISA using a cocktail of recombinant proteins from Onchocerca volvulus to evaluate the specificity of the test in an endemic M. ozzardi region. No cross-reactions between M. ozzardi-infected individuals and recombinant O. volvulus proteins were detected, thus providing information on the secure use of this particular cocktail in areas where these parasites are sympatric.     

Effects of Temperature,Shoot Age,and Medium on Gall Induction by Subanguina picridis in Vitro

The influence of temperature, shoot age, and medium on gall induction by Subanguina picridis on Russian knapweed (Acroptilon repens) was examined in vitro. The optimal temperature for gall formation was 20 C. Gall induction was delayed as the temperature decreased, and decreased as shoot age increased. Bud primordia (0-day-old shoots and 5-day-old shoots) with an average length of 4.2 mm and 7.9 mm were the most suitable tissues for nematode development and gall formation. Gall formation was more effective on B5G medium than on MSG. Young shoots under slow growth were most suitable for mass rearing of S. picridis.     

Cultivation of the Pine Wilt Nematode, Bursaphelenchus xylophilus,in Axenic Culture Media

The pine wilt nematode, Bursaphelenchus xylophilus, has been cultured axenically in vitro on soy peptone/yeast extract or modified Caenorhabditis medium supplemented with cholesterol and hemoglobin. Although growth, development and reproduction were best in soy peptone/yeast extract medium, satisfactory population size increases were observed in the chemically defined Caenorhaditis medium.     

In Vitro Embryo Explant Cultures of Peanut to Evaluate Resistance to Ditylenchus destructor

Population densities of D. destructor on embryo explants of 22 peanut genotypes grown in vitro were compared with those in roots and seeds of the same genotypes grown in the greenhouse. During the first 8 weeks after inoculation, the optimum incubation period was 6 weeks for maximum reproduction of Ditylenchus destructor on embryo explants of peanut (Arachis hypogaea L. cv. Sellie) inoculated with 250 nematodes at 25 C. Nematode numbers increased 17-fold. Deletion of MnSO₄ H₂O and a higher KH₂PO₄ concentration in the medium resulted in higher nematode reproduction. Resistance or susceptibility to D. destructor was observed in seeds of several genotypes but was not matched by differences in host suitability in roots. The results indicate that the factor for resistance or susceptibility to D. destructor is synthesized in the seeds of peanut but is not translocated to the roots. Use of embryo explant cultures of peanut as a rapid method to evaluate resistance to D. destructor did not work under the conditions described.     

Meloidogyne incognita Inoculum Source Affects Host Suitability and Growth of Yellow Nutsedge and Chile Pepper

Meloidogyne incognita (Mi) reproduction and host plant responses in chile pepper (Capsicum annuum) and yellow nutsedge (Cyperus esculentus = YNS) to three sources of inoculum obtained by rearing a single Mi population on chile, YNS, and tomato were evaluated in two factorial greenhouse experiments. The interactive effects of Mi inoculum source and crop-weed competition were determined. In the absence of YNS competition, chile growth was reduced less by Mi inoculum from chile than by inoculum from YNS or tomato. When YNS was present, chile root weight was not affected and shoot weight increased with Mi initial inoculation, regardless of inoculum source. Chile plants inoculated with Mi from tomato exhibited double the nematode reproduction observed with inoculum from chile or YNS. With chile present, Mi reproduction on YNS was nearly three times greater with inoculum from tomato, but reproduction was similar among inoculum sources when chile was absent. Reductions in YNS root mass due to competition from chile failed to reduce the total number of Mi eggs produced on YNS plants. Differences in total Mi reproduction among inoculum sources were not attributable to differences in root growth or plant competition. This study illustrates the influence of Mi-YNS interactions and previous hosts on severity of Mi infection.     

Sterile Culture of Rotylenchulus reniformis on Tomato Root with Gellan Gum as a Supporting Medium

Rotylenchulus reniformis was repeatedly propagated in sterile excised tomato roots growing on modified White''s medium with gellan gum as the support. Gellan gum provided an optically clear support medium that could be liquified by adding 5 mM disodium ethylenediaminetetraacetate (EDTA) to facilitate nematode extraction. Liquefaction of the gellan-gum medium by EDTA allowed efficient recovery of eggs and vermiform stages of R. reniformis. Extraction efficiency was quantified with Radopholus similis as a test organism. The efficiency of extracting R. similis from the gellan gum did not vary with the concentrations of EDTA tested.     

Interaction between Meloidogyne incognita and Hoplolaimus columbus on Davis Soybean

Greenhouse and laboratory experiments were performed to determine if an interaction exists between Meloidogyne incognita and Hoplolaimus columbus on Davis soybean. Greenhouse tests were performed with three population levels of M. incognita and H. columbus (0, 1,500, 6,000/1.5-liter pot) separately and in all combinations. Dry root weight (DRT) declined nonlinearly and dry shoot weight (DST) declined linearly with respect to increasing initial populations of M. incognita and H. columbus. When the two nematode species were added to the soil together, the amount of DRT and DST suppression by one species was dependent on the initial level of the concomitant species. The final root population of M. incognita or H. columbus declined linearly with increasing initial population density of the concomitant species. H. columbus suppressed M. incognita populations in the soil nonlinearly, but M. incognita had no effect on H. columbus.     

Interpreting Upland Rice Yield and Pratylenchus zeae Relationships: Correspondence Analyses

Correspondence analyses were used to explore the relationships between yield and populations of Pratylenchus zeae in an upland rice field and in a greenhouse experiment. Initial soil (Pi) and final root (Pf) population densities of P. zeae, and yield (Y) of rice cv. UPL Ri5 were determined at 490 spots in the field. Very low Y was linked to very high Pf. Low Y was linked to medium or high Pi and medium Pf. Medium to very high Y were clustered with undetectable Pi and very low or high Pf. All yield levels were independent of very high Pi. In the greenhouse experiment where seven nematode inoculum levels and three fertilizer levels were evaluated, low Y was associated with medium or high Pf and high Y with high or low Pf. The analyses indicated that nematode-yield interaction involved a complex, dynamic process, in which the root-carrying capacity probably was a determining factor. Correspondence analysis, which does not require assumptions on the shape of nematode population-yield relationships or on variable distributions, revealed meaningful associations in these complex data sets.     

Taxonomic revision of the genus Callimerus Gorham s. l. (Coleoptera,Cleridae). Part I. latifrons species-group

The latifrons species-group (=Brachycallimerus sensu Chapin 1924, Corporaal 1950; = flavofasciatus-group sensu Kolibáč 1998) of Callimerus Gorham is redefined and revised. Five species are recognized including one new species Callimerus cacuminis Yang & Yang sp. n. (type locality: Yunnan, China). Callimerus flavofasciatus Schenkling, 1902 is newly synonymized with Callimerus latifrons Gorham, 1876. Callimerus trifasciatus Schenkling, 1899a is transferred to the genus Corynommadius Schenkling, 1899a. Callimerus gorhami Corporaal, 1949 and Callimerus pallidus Gorham, 1892 are excluded from the latifrons species-group (their assignment to a species-group will be dealt with in a subsequent paper). A key to species of the latifrons species-group is given and habitus of each type specimen, male terminalia, and other diagnostic characters are illustrated.     

Plant-parasitic Nematode Acetylcholinesterase Inhibition by Carbamate and Organophosphate Nematicides

The sensitivity of acetylcholinesterases (ACHE) isolated from the plant-parasitic nematodes Meloidogyne arenaria, M. incognita, and Heterodera glycines and the free-living nematode Caenorhabditis elegans to carbamate and organophosphate nematicides was examined. The AChE from plant-parasitic nematode species were more sensitive to carbamate inhibitors than was AChE from C. elegans, but response to the organophosphates was approximately equivalent. The sulfur-containing phosphate nematicides were poor inhibitors of nematode acetylcholinesterase, but treatment with an oxidizing agent greatly improved inhibition. Behavioral bioassays with living nematodes revealed a poor relationship between enzyme inhibition and expression of symptoms in live nematodes.