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Ralf-Udo Ehlers and Richou Han

Abstract

Mixed culture filtrates of Photorhabdus luminescens isolated from Heterorhabditis bacteriophora H06 and from H. indica LN2 had a toxic effect on axenic H. bacteriophora H06 dauer juveniles. Single culture filtrates had no effect. When one filtrate originated from a secondary phase culture, the toxic effect was lost. Heat treatment of one of the filtrates to 80°C destroyed the effect. The toxin is probably synthesized de novo after mixing the culture supernatants of these two P. luminescens symbionts. Dilution of the filtrate reduced the effect, and it was lost when P. luminescens was cultured for more than two days. Steinernema carpocapsae A24 was not affected by the mixture, and was affected only by the filtrate of primary phase P. luminescens H06. The toxic effect was recorded also when axenic dauer juveniles of H. bacteriophora were inoculated into a mixed bacterial culture of H06 and LN2. Inoculating monoxenic dauer juveniles of H. bacteriophora H06 into P. luminescens LN2 or into mixtures containing LN2 bacteria resulted in significant dauer juvenile mortality. These manifestations of the interaction of bacteria to produce toxic effects on the non-symbiotic nematode (trans-specific activity) may have an impact on competitive interactions when one insect host is infected by different nematode species. Eine transspezifische nematizide Aktivitat von Photorhabdus luminescens - Eine Mischung der Kulturfiltrate des Bakteriums Photorhabdus luminescens, isoliert aus Heterorhabditis bacteriophora H06 mit dem Filtrat des Symbionten aus H. indica LN2 wirkt toxisch auf axenische Dauerlarven von H. bacteriophora H06. Die einzelnen Kulturfiltrate zeigten keine Wirkung. Sofern ein Filtrat einer Sekundarformkultur entnommen wurde, konnte kein Effekt erzielt werden. Eine Hitzebehandlung bei 80°C zerstorte die Wirkung. Das Toxin wird wahrscheinlich de novo synthetisiert in dem Moment, in dem die Kulturfiltrate dieser P. luminescens Symbionten gemischt werden. Eine Verdunnung der Filtrate reduzierte die Wirkung. Sie ging ganz verloren, wenn P. luminescens langer als zwei Tage kultiviert wurde. Die Mischung hatte keine Wirkung auf axenische Steinernema carpocapsae. Dieser Nematode wurde nur durch Filtrate der Primarform von P. luminescens H06 abgetotet. Die toxischen Effekte wurden auch festgestellt, wenn axenische Dauerlarven von H. bacteriophora in Gemische von H06 und LN2 Bakterienkulturen inokuliert wurde. Bei Inokulation von monoxenischen Dauerlarven von H. bacteriophora H06 in P. luminescens LN2 oder in Gemische, die LN2 enthielten, wurde ebenfalls eine gesteigerte Mortalitat festgestellt. Dieses Deutlichwerden von Wechselbeziehungen zwischen den Bakterien bei Erzeugung toxischer Effekte auf den nicht-symbiontischen Nematoden (trans-spezifische Aktivitat) konnte eine Auswirkung auf die Konkurrenz bei Koinfektion eines Wirtsinsekts mit zwei Nematodenarten haben.

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Wenxiu Guo, Xun Yan and Richou Han

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.

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Xun Yan, Xiandong Wang, Richou Han and Xuehong Qiu

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.

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Xun Yan, Yinying Lin, Zhenmao Huang and Richou Han

Summary

The biological and biocontrol traits of two entomopathogenic nematode isolates, Steinernema pakistanense 94-1 (Sp94-1) and Heterorhabditis indica 212-2 (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) × 105 and 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, while S. carpocapsae All, a commercial strain, was more resistant to osmotic pressure, desiccation, cold treatment and hypoxia than the other tested isolates. Hi212-2 suppressed the Phyllotreta striolata larvae when applied at 1.5 × 109 IJ ha−1 or higher concentrations, while Sp94-1 suppressed the P. striolata larvae 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 the P. striolata larvae in the field.

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Xiuling Liu, Li Cao, Xiaofen Liu, Richou Han and Xuehong Qiu

Abstract

Xenorhabdus and Photorhabdus bacteria are symbionts of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis, respectively. To determine the nutrient potential of these bacteria for a free-living nematode, Panagrellus redivivus, a promising food source for first-feeding fish and crustacean, sterile first-stage juveniles (J1) of P. redivivus were fed on various isolates of Xenorhabdus and Photorhabdus bacteria in liquid cultures. Most of the tested bacterial isolates did not support the growth of P. redivivus. However, four of the Xenorhabdus isolates (X. nematophila All, X. bovienii T319, X. beddingii X-7 and X. poinarii KG) provided nutrients for the production of these nematodes in a liquid medium. Two Xenorhabdus isolates (X. beddingii X-7 and X. poinarii KG) even supported mass production of the nematode in a sponge medium, with yields comparable to those with yeast strains. This is the first report that Xenorhabdus bacteria can function as a nutrient source for mass production of nematodes other than their usual symbiotic partners.

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Richou Han, John Jones, Xun Yan, Shulong Chen, Patrick De Clercq and Maurice Moens

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.

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Shahina Fayyaz, Xun Yan, Lihong Qiu, Richou Han, Mehreen Gulsher, Tabassum Ara Khanum and Salma Javed

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.

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Juan Ma, Shulong Chen, Xiuhua Li, Richou Han, Hari Bahadur Khatri-Chhetri, Patrick De Clercq and Maurice Moens

A new species of Steinernema was isolated from shrub soils in Liaoning province during a survey for entomopathogenic nematodes in north China. This nematode was obtained by the insect-baiting technique using last instar larvae of Galleria mellonella (Lepidoptera: Pyralidae). This new species is described herein as S. tielingense n. sp. It belongs to the ‘feltiae-kraussei-oregonense’ group and is characterised by infective third-stage juvenile (IJ) with a body length of 915 (824-979) μm, distance from head to excretory pore of 69 (64-73) μm, tail length of 81 (74-85) μm, ratio E=88 (85-94)%, lateral field with eight ridges at mid-body, first generation male spicule 88 (79-98) μm and gubernaculum 61 (49-70) μm long, and first generation female with a vulval protrusion and ratio D=41 (32-49)%. The new species distinctly differs from the related species S. kraussei, S. silvaticum, S. oregonense and S. cholashanense in the different number of ridges in the lateral fields and hyaline tail length as % of total tail length of IJ and male body length and distance from head to excretory pore. Cross hybridisation tests showed that these species were reproductively isolated. The sequences analyses of the internal transcribed spacers (ITS) and D2-D3 regions of the ribosomal DNA confirm this to be a new species.

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Juan Ma, Juan Ma, Shulong Chen, Juan Ma, Shulong Chen, Patrick De Clercq, Juan Ma, Shulong Chen, Patrick De Clercq, Lieven Waeyenberge, Juan Ma, Shulong Chen, Patrick De Clercq, Lieven Waeyenberge, Richou Han, Juan Ma, Shulong Chen, Patrick De Clercq, Lieven Waeyenberge, Richou Han and Maurice Moens

During a survey for entomopathogenic nematodes in northern China, a new species of Steinernema was isolated from soil samples collected from Xinbin county, Liaoning province. This nematode was obtained by the insect-baiting technique using last instar larvae of Galleria mellonella. It is described herein as S. xinbinense n. sp. The nematode can be separated from other described species of the group by morphological and morphometric characteristics of the different stages and by characterisation and phylogeny of DNA sequences of the D2D3 domain of the LSU or ITS regions of rDNA. This new species is characterised by the following morphological characters: infective third-stage juvenile with a body length of 694 (635-744) μm, distance from head to excretory pore of 51 (46-53) μm, tail length of 73 (61-81) μm, E = 71 (65-78)%, presence of eight unevenly spaced and developed ridges in middle lateral field (i.e., nine lines). First generation male with well curved, yellowish spicules 56 (49-62) μm long and gubernaculum 35 (30-41) μm long, small mucron mostly present, first generation female with protruding vulva, tail conical with one or two small mucrons and D = 45 (41-50)%. Cross hybridisation tests with S. tielingense, S. kraussei, S. feltiae and S. hebeiense showed that this species was reproductively isolated. The analyses of ITS-rDNA and D2D3 sequence confirm that the studied nematode isolate is a valid new species belonging to the ‘feltiae-kraussei-oregonense’ group.