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SummaryThe biological and biocontrol traits of two entomopathogenic nematode isolates,Steinernema pakistanense94-1 (Sp94-1) andHeterorhabditis indica212-2 (Hi212-2), were evaluated. The highest yield of infective juveniles (IJ) in monoxenic sponge culture system for Sp94-1 and Hi212-2 was 3.52 (± 0.45) × 105and 7.08 (± 0.11) × 105 IJ g−1, respectively. The optimum storage temperature was 25°C for Sp94-1 and 14°C for Hi212-2. Sp94-1 showed greater tolerance to heat exposure and UV radiation, whileS. carpocapsaeAll, a commercial strain, was more resistant to osmotic pressure, desiccation, cold treatment and hypoxia than the other tested isolates. Hi212-2 suppressed thePhyllotreta striolatalarvae when applied at 1.5 × 109 IJ ha−1or higher concentrations, while Sp94-1 suppressed theP. striolatalarvae only when applied at 4.5 × 109 IJ ha−1. Our study indicates the possibility of commercialisation of the EPN isolates, and further confirms their efficacy against theP. striolatalarvae in the field.

The effects of carriers, temperatures, concentrations of the infective juveniles (IJ) and a fungicide on the survival and infectivity of five nematode species, Steinernema carpocapsae, S. feltiae, S. longicaudum, Heterorhabditis bacteriophora and H. indica, were evaluated to establish the adapted formulations for these nematodes. Vermiculite and humus were good carriers for the storage of the three Steinernema species, with more than 90% IJ survival after 120 days at 5°C, 80 days at 15°C and at least 20 days at 25°C, and 90% survival for the storage of H. bacteriophora after 10 days at 5°C and 15°C. After 10 days at 25°C, ca 80% IJ survival was recorded for H. bacteriophora and H. indica. Although ca 90% IJ survival was found after 10 days at 15°C for H. indica, this species did not tolerate low temperature, with survival less than 40% after 10 days at 5°C. The ratios of the IJ and the carriers in the ranges of 1:0.8-1:1.2 (w/w) did not significantly influence the survival of all nematode species. The vermiculite formulation containing a fungicide Proxel GXL at concentrations of 0.1% and 0.2% increased the survival of two Heterorhabditis species. Heterorhabditis bacteriophora and H. indica could be stored for 60 and 40 days, respectively, at 15°C in aerated water with 90% IJ survival, compared with the vermiculite formulation. The tested formulations did not significantly influence the infectivity of the IJ from the formulations with IJ survival more than 80%. The results provide alternative formulation methods for the commercial storage of these beneficial nematodes.

A new species of Steinernema is described herein as S. bifurcatum n. sp. It is characterised by the presence of a male gubernaculum that is bifurcate at both proximal and distal ends, a key diagnostic feature. Steinernema bifurcatum n. sp. belongs to the bicornutum group by having two horn-like structures on the labial region of the infective juvenile (IJ). It can be recognised by IJ body diam. = 22 (20-24) μm, pharynx = 114 (102-134) μm, ratio a = 24 (22-25) and D% = 39.7 (33-47). The new species can be further recognised by the male characters of D% = 48 (42-58), and genital papillae = 23 (22 + 1) in number and, for the first generation female, excretory pore = 75.7 (60-108) μm, pharynx = 174 (158-200) μm and tail length = 43.2 (38-60) μm. Steinernema bifurcatum n. sp. is distinguished from all other members of the bicornutum group by the presence of a gubernaculum which is bifurcated at both proximal and distal ends in first generation males. On the basis of genital papillae number (22 + 1) it is close to S. abbasi, S. ceratophorum and S. pakistanense, but can be distinguished by morphometrics of IJ and adults. IJ of S. bifurcatum n. sp. can be differentiated from those of S. ceratophorum and S. pakistanense by the smaller body length of 460-590 μm. Steinernema bifurcatum n. sp. differs from S. abbasi by morphological characters of IJ, male and female stages. The IJ can be distinguished by D% (33-47), body diam. (20-24) μm, b value (3.8-5.6) and pharynx length (102-134) μm. The second generation male differs by GS ratio (0.29-0.45) and the mucronate tail. The first generation female is distinguished by a protruding vulva and no postanal swelling. Analysis of ITS rDNA (824 bp), D2-D3 (880 bp) and mt DNA region (507 bp) sequences confirm that the studied nematode isolate represents a valid new species, the combination of molecular and morphological features indicating that it belongs to Clade IV, the bicornutum group.

Entomopathogenic nematodes (EPN) have good application prospects for the control of the black cutworm, Agrotis ipsilon. In the present study, the effects of isolate, exposure rate, temperature and larval stage on EPN infectivity to A. ipsilon were evaluated. Results of in vitro tests showed that Steinernema carpocapsae Mex and Heterorhabditis indica LN2 were the most virulent and promising species, causing 80.0 and 83.3% mortality, respectively, to the third instar larvae at 25°C and 72 h after infection. Mortality of A. ipsilon caused by the nematodes was significantly affected by EPN exposure rate, temperature and the larval stage. Both S. carpocapsae and H. indica caused greater mortality to the third instar of A. ipsilon at 25°C than at 15, 20 and 30°C. Both EPN isolates also caused higher mortality to the second instar than to the third and fourth instar larvae of A. ipsilon. The field trials of EPN for the control of A. ipsilon also showed that S. carpocapsae Mex and H. indica LN2 reduced the damage caused by A. ipsilon and increased the cabbage yield when compared with the control. Both EPN isolates showed better control effects than cyfluthrin and Bacillus thuringiensis, indicating that these two EPN isolates could be used for sustainable control of A. ipsilon in vegetable fields in China.

Abstract A solution consisting of a mixture of glycerol and fortified artificial seawater was used to induce partial anhydrobiosis at 15°C in different strains of Steinernema carpocapsae. All the strains tested were tolerant to the osmotic solution and the survival and infectivity of the nematodes were not influenced. The osmotic treatment greatly improved heat tolerance of all the tested strains but the heat tolerance of S. carpocapsae MR7 was significantly poorer than that of the other strains. This method could be used to induce S. carpocapsae into partial anhydrobiosis and thereby improve storage of the nematodes. Expression patterns of stress-related genes after osmotic treatment were compared in a heat tolerant strain (All) and a more heat sensitive strain (MR7) after induction of anhydrobiosis. Differences in gene expression after induction of anhydrobiosis between strain MR7 and All were observed that may be related to differences in subsequent heat tolerance.