Development of the one-on-one quality assessment assay for entomopathogenic nematodes.

The one-on-one bioassay was developed using Steinernema carpocapsae (All) nematodes against the wax moth larva, Galleria mellonella. The assay was used to develop and compare virulence profiles of both in vitro- and in vivo-produced nematodes and to provide a quality assessment 'standard' for in vitro-produced nematodes. The bioassay was subsequently used to develop virulence profiles for Steinernema carpocapsae (UK), S. feltiae (UK), S. feltiae (R1.5), S. feltiae (SN), S. glaseri (NJ-43), and S. riobrave (RGV). These profiles are unique for each species and isolate and are used as a standard of virulence in routine quality assessment of nematodes produced in liquid fermentation.

Influence of production and bioassay methods on infectivity of two ambush foragers (Nematoda: steinernematidae)

The influence of production and bioassay methods on infectivity of two ambush foragers, Steinernema carpocapsae and Steinernema scapterisci, was evaluated. Both species were mass-produced in vitro in liquid culture and in vivo using live insects: S. carpocapsae in last instar wax moth Galleria mellonella larvae and S. scapterisci in adult house crickets Acheta domesticus. Infectivity was assessed at 28 degreesC against last-instar G. mellonella larvae using a filter-paper bioassay and a newly developed "sand-well" bioassay. The infectivity of S. carpocapsae was not influenced by the method of production or bioassay, whereas the infectivity of S. scapterisci was influenced by both factors. Both in vivo and in vitro produced S. carpocapsae caused >60% larval mortality at one nematode per larva (1:1) in the filter-paper bioassay, but S. scapterisci elicited less than 10% mortality. At 50 nematodes per larva, in vitro S. scapterisci caused 41.7% mean larval mortality in the filter-paper bioassay, whereas in vivo S. scapterisci elicited only 28.5% mortality. The replacement of filter paper with a 2.5-mm-deep layer of sand (termed sand well) resulted in 2.5-fold increase in infectivity of S. scapterisci. In the new sand-well procedure, 15 S. scapterisci per larva (15:1) caused an overall mean larval mortality of 47.5% and the pattern of mortality showed a normal distribution. The infectivity of S. carpocapsae was not different in the 1:1 filter paper or 1:1 sand-well bioassay. These results demonstrate that nematode infectivity could be strongly influenced by both the production and bioassay methods, and there are no universal assays even when nematodes have similar foraging strategies. Copyright 1999 Academic Press.

Virulence of entomopathogenic nematodes to the western masked chafer Cyclocephala hirta (Coleoptera: Scarabaeidae).

Predictability is a key challenge in biological control of white grubs with entomopathogenic nematodes. Most field test failures have been attributed to the use of inappropriate nematode strains. We evaluated several species and strains of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) against chafer Cyclocephala hirta in a soil and pot bioassay at 25 degrees C. The NJ65 strain of Steinernema glaseri, isolated from New Jersey, outperformed all other steinernematid and heterorhabditid nematodes, resulting in a 76.5% larval mortality within 3 d of treatment at 125 nematodes per larva. After 6 days of treatment, 4 strains of S. glaseri (NJ21, NJ29, NJ42, and NJ65) achieved 100% larval mortality. Other strains that caused > 80% larval mortality after 6 d of treatment included NJ32, NJ40, and NJ41 of S. glaseri, and Chino Hill, Merced, and Nebraska strains of Heterorhabditis sp. Steinernema anomali (Ryazan), Steinernema kushidai (Hamakita), Heterorhabditis megidis (HO1), and H. bacteriophora (HP88) caused only 45, 55, 60, and 66.7% larval mortality, respectively. Steinernema feltiae (Argentina strain) caused only 16% larval mortality, and Steinernema carpocapsae (All and Mexican strains) and Steinernema scapterisci (Colon strain) were nonpathogenic to C. hirta. Steinernema riobravis caused no larval mortality at 25 degrees C, but inflicted 45-71% mortality at 30 degrees C. Our studies indicate that S. glaseri and Heterorhabditis spp. are most virulent among entomopathogenic nematodes toward C. hirta larvae and certain strains of S. glaseri are superior to Heterorhabditis spp.

Suitability of 30 Agricultural Products and By-Products as Nutrient Sources for Laboratory Production of Bacillus thuringiensis subsp. aizawai (HD133)

Bacillus thuringiensis subsp. aizawai (HD133) was grown in culture media in which dextrose was a common carbon source and 30 different agricultural products and by-products were tested as the main nitrogen sources. These products included legumes, cereals, animal proteins, leaf proteins, yeasts, oilseeds, tubers, and casamino acid. Of the 30 products tested, cottonseed meal, defatted soy flour, and corn gluten meal were the most efficient substrates for the production of spore-crystal biomass and endotoxin potency. The carbohydrate/nitrogen ratios for these additives ranged from 0.3 to 0.5 and the glutamic acid content of their proteins from 9.2 to 16.0%. There was no close relationship between the estimates of the amounts of endotoxin produced and the potency of the product when fed to bertha armyworm, Mamestra configurata.

Mechanisms of specificity of association between the nematode Steinernema scapterisci and its symbiotic bacterium.

We suggest a new mechanism for the maintenance of specificity of the association between the entomopathogenic nematode Steinernema scapterisci and its symbiotic bacteria. We evaluated the development and reproduction of infective and non-infective juvenile S. scapterisci in monoxenic combinations with its symbiotic bacteria, Xenorhabdus sp. 'S' and with the bacterial symbiont of Steinernema carpocapsae and Steinernema riobravis. Although development of non-infective stages occurred on all Xenorhabdus spp., the development of infective juveniles to the 4th stage ('dauer' recovery) was significantly delayed and reduced with X. nematophilus and Xenorhabdus sp. 'R', the bacterial symbionts of S. carpocapsae and S. riobravis, respectively. 'Dauer' recovery improved significantly when the cultures of X. nematophilus and Xenorhabdus sp. 'R' were supplemented with cell-free filtrates from Xenorhabdus sp. 'S'. The infective juvenile S. scapterisci produced in all 3 cultures were virulent to Galleria mellonella larvae, confirming successful retention of Xenorhabdus from other steinernematids in their intestine. In fact, S. scapterisci infective juveniles containing X. nematophilus or Xenorhabdus sp. 'R' were more virulent to G. mellonella than those containing their natural symbiont, Xenorhabdus sp. 'S'. We believe that this is the first demonstration of the symbiont-specific exit of infective juveniles from the 'dauer' phase which represents the finest level of specificity of bacteria-nematode association. This is also the first report of successful isolation of the natural symbiont of S. scapterisci.