Advance of entomopathogenic nematodes

This paper summarized the history and present condition of studying and utilizing entomopathogenic nematodes at home and abroad, expounded its taxonomy, life cycle and the mechanism with symbiotic bacteria killing host insect. Taxonomy, mycelial form, pathogenic function and anti-bacteria function of symbiotic bacteria were introduced. Production and utilization of entomopathogenic nematodes, the characteristic genetic improvement by use of biological engineering technology, as well as the existing problem and applying foreground were also discussed.

Pathogenicity of bacterium, Xenorhabdus nematophila isolated from entomopathogenic nematode (Steinernema carpocapsae) and its secretion against Galleria mellonella larvae

The entomopathogenic bacterium, Xenorhabdus nematophila was isolated from the hemolymph of Galleria mel- lonella infected with Steinernema carpocapsae. The bacterial cells and its metabolic secretions have been found lethal to the Galleria larvae. Toxic secretion in broth caused 95% mortality within 4 d of application whereas the bacterial cells caused 93% mortality after 6 d. When filter and sand substrates were compared, the later one was observed as appropriate. Similarly, bacterial cells and secretion in broth were more effective at 14% moisture and 25 °C temperature treatments. Maximum insect mortality (100%) was observed when bacterial concentration of 4×106 cells/ml was used. Similarly, maximum bacterial cells in broth (95%) were penetrated into the insect body within 2 h of their application. However, when stored bacterial toxic secretion was applied to the insects its efficacy declined. On the other hand, when the same toxic secretion was dried and then dissolved either in broth or water was proved to be effective. The present study showed that the bacterium, X. nematophila or its toxic secretion can be used as an important component of integrated pest management against Galleria.

Microbial control of diamondback moth, Plutella xylostella L. （Lepidoptera： Yponomeutidae） using bacteria （Xenorhabdus nematophila） and its metabolites from the entomopathogenic nematode Steinernema carpocapsae

Cells and cell-free solutions of the culture filtrate of the bacterial symbiont, Xenorhabdus nematophila taken from the entomopathogenic nematode Steinernema carpocapsae in aqueous broth suspensions were lethal to larvae of the diamondback moth Plutella xylostella. Their application on leaves of Chinese cabbage indicated that the cells can penetrate into the insects in the absence of the nematode vector. Cell-free solutions containing metabolites were also proved as effective as bacterial cells suspension. The application of aqueous suspensions of cells of X.nematophila or solutions containing its toxic metabolites to the leaves represents a possible new strategy for controlling insect pests on foliage.

Biocontrol potential of entomopathogenic nematode,Heterorhabditis indica against pink bollworm,Pectinophora gossypiella(Saunders)(Lepidoptera:Gelechiidae)

Background The emergence of pink bollworm(PBW),Pectinophora gossypiella(Saunders)(Lepidoptera:Gelechiidae),in cotton due to Bt resistance and concealed feeding habit has created a need for alternative,eco-friendly,and cost-effective control methods.This study aimed to evaluate the bio-efficacy and reproductive potential of two native strains of entomopathogenic nematodes(EPNs),Heterorhabditis indica,namely CICR-HI-CL and CICR-HI-MN,against PBW larvae and pupae under in-vitro conditions.Results The larval assay revealed that strain CICR-HI-CL exhibited higher potency than strain CICR-HI-MN against 2nd,3rd,and 4thinstar larvae,with median lethal dose(LD50)values of 5.45,4.45,and 4.60 infective juveniles(IJs)per larva,respectively.In case of pupal bioassay,both EPN strains demonstrated greater virulence when applied directly(LD50values:29.65 and 73.88 IJs per pupa for strains CICR-HI-CL and CICR-HI-MN,respectively)compared to soil application(147.84 and 272.38 IJs per pupa).Both EPN strains successfully penetrated and reproduced on 4thinstar larvae,resulting in maximum production of 19.28 and 20.85 lakh IJs per larva in the next generation when inoculated at 30 IJs per larva.Conclusion The present study has generated useful information on the virulence and reproductive potential of two strains of EPN H.indica(CICR-HI-CL and CICR-HI-MN)against PBW,a dreaded pest of cotton.Higher virulence and reproductive potential of EPN strains demonstrated their ability to multiply,sustain and perpetuate on larval and pupal stages of PBW.The knowledge generated will help formulate effective management strategies for PBW with the inclusion of EPN as a potential biological control candidate.The soil-dwelling life stages viz.,last instar hibernating larvae and pupae of PBW can be the ideal weak links to make a successful use of H.indica for sustainable management of PBW in the cotton ecosystem.However,before taking these EPN strains to field for managing PBW,detailed studies investigating their biocontrol potential against PBW under field conditions are needed.

Interactions between Entomopathogenic Nematodes and Entomophagous Insects

The objective of this review article is to clarify the different interactions between entomopathogenic nematodes, as bio-control agents, against other bio-control agents (parasitoids and predators) proposed by the researchers. Thus, it gives clear information concerning the potential of combining them as a part of Integrated Pest Management (IPM) programs against insect pests. Some laboratory studies showed that the treatment of predacious insects by entomopathogenic nematodes (EPNs) at different concentrations can infect and kill the treated larva, nymphs and adults of predators. The percentages of mortality were found to be high (up to 100%), moderate (15% - 35%) or low (3% - 7%). Other studies revealed the resistant of treated predators to nematode-infection. Some predators that were offered infected prey avoid feeding on such prey and, in contrast, the soil predators (ants and mites) consumed the offered cadavers as well as the infective juvenile of the nematodes and did not show any detrimental effects. Mostly, parasitoids cannot complete their development inside or on nematode-infected hosts if parasitism occurs before or early after infection. The parasitoid females may avoid laying eggs in the infected hosts or cannot discriminate between healthy and infected hosts. A field study demonstrated that applying EPNs combined with the predator, Labidura riparia significantly reduced the population of the target pest compared to the nematode or the predator alone. Also, two field experiments indicated that the combination of parasitoids and nematodes can be successful for insect pest management.

Susceptibility of Economic Dipteran Fruit Flies to Entomopathogenic Nematodes

The present review article demonstrates laboratory and field evaluations of entomopathogenic nematodes(EPNs)against different developmental stages of fruit flies.The virulence of the EPNs differed clearly even on the same insect species and/or by the same nematode species.Such differences might be attributed to some reasons such as the method of treatment as well as the concentrations of the tested nematodes.Fruit flies are among the most important insect pests infesting vegetables and fruits causing considerable losses in the yields worldwide.In laboratory studies,the tested nematodes proved to be highly virulent to larvae as percentage of mortality may reach 100%.As for treated pupae,at different ages,the results are variable and controversially;some studies revealed their moderate or high susceptibility to nematode infection and others indicated low susceptibility or resistance to infection.Treated adults,or those emerged from treated larvae or pupae,are also susceptible to infection.In semi-field and field trials,EPNs proved to be successful for reducing the populations of some fruit flies with up to 85%at concentrations not less than 100 infective juveniles(IJs)/cm^2 of soil.However,the field applications of commercial EPNs have been recommended to be 2.5-5 x 10^9 IJs/ha(25-50 IJs/cm^2 of soil).

The integrated use of chemical insecticides and the entomopathogenic nematode, Steinemema carpocapsae （Nematoda： Steinernematidae）, for the control of sweetpotato whitefly, Bemisia tabaci （Hemiptera： Aleyrodidae）

The integration of chemical insecticides and infective juveniles of the entomopathogenic nematode Steinernema carpocapsae （Wesier） （Nematoda： Steinernematidae）, to control second instars of the sweetpotato whitefly, Bemisia tabaci Gennadius （Hemiptera： Aleyrodidae） was investigated. Using a sand bioassay, the effects of direct exposure of S. carpocapsae for 24 h to field rate dilutions of four insecticides （spiromesifen, thiacloprid, imidacloprid and pymetrozine） on infectivity to Galleria mellonella larvae were tested. Although all chemicals tested, except spiromesifen, produced acceptable nematode infectivity rates, they were all significantly less than the water control. The effect of insecticide treatment （dry residues of spiromesifen, thiacloprid and pymetrozine and soil drench of imidacloprid） on the efficacy of the nematode against B. tabaci was also investigated. Nematodes in combination with thiacloprid and spiromesifen gave higher B. tabaci mortality （86.5% and 94.3% respectively） compared to using nematodes alone （75.2%） on tomato plants. There was no significant difference in B. tabaci mortality when using the chemicals imidacloprid, pymetrozine and spiromesifen in conjunction with nematodes compared to using the chemicals alone. However, using thiacloprid in combination with the nematodes produced significantly higher B. tabaci mortality than using the chemical alone. The integration of S. carpocapsae and these chemical agents into current integrated pest management programmes for the control of B. tabaci is discussed.

Storage of osmotically treated entomopathogenic nematode Steinernema carpocapsae

The infective juveniles （IJs） ofSteinernema carpocapsae ‘All＇ were osmotically stressed by a mixture of ionic （fortified artificial seawater） and non-ionic （3.2 mol/L glycerol） solutions to establish a method for osmotic storage of entomopathogenic nematodes. Seven combinations （termed solution A to G） with different proportions of these two solutions were tested, with sterile extra pure water （sepH20, termed solution H） as a control. The mortality of the IJs at a concentration of 5×10^5 IJ/mL in the solutions A to G, and H were 13.2%, 16.2%, 16.7%, 13.5%, 25.2%, 31.6%, 44.6%, and 1.0%, respectively, after 21 days storage at 25℃. Most of the IJs shrunk and stopped motility after 6-9 hours incubation at 25℃ in solutions A to D. Based on the results, solutions A to D and H were chosen to further test the osmotic survival of the IJs at different IJ concentrations （5× 10^5, 2.5×10^5, 2 000 IJ/mL） and incubation temperature （30℃, 25℃, 10℃）. The resulting IJs were exposed to a high temperature assay （45℃ for 4 h, HTA）. Osmotically stressed IJs showed improved heat tolerance. The mortality of the Lls increased with the increasing concentrations of the test Lls and the storage temperatures after exposing to the HTA. More than 88.4%, 62.3% or 2.4% of the treated IJs died at the above three IJ concentrations, respectively. At the three IJ concentrations （2 000 IJs/mL, 2.5×10^5 IJs/mL or 5 ×10^5 IJs/mL）, the highest mortality was recorded in solution D （11.6%, 85.9% or 98.0%, respectively）, and the lowest mortality in solution B （2. 4%, 62.3% or 86.6%, respectively）. No untreated IJs survived after the heat treatment. During 42 days storage at 10℃, the IJs mortality in the solutiors A to D and H were 7.19%, 5.97%, 4.41%, 4.34%, and 4.34% respectively, and showed no significant differences. In conclusion, osmotic treatment of the IJs of S. carpocapsae ‘ All＇ in a mixture of ionic and non-ionic solutions enhances the heat tolerance. The mortality of the IJs after HTA increased with the increasing concentrations of the test IJs and the storage temperatures after exposure to the HTA. The result is promising for the osmotic storage of the entomopathogenic nematodes.

Pseudomonas oryzihabitans Suppresses Damage Caused by Root-knot Nematode Meloidogyne javanica on Tomato

Agricultural research made in recent years has found that many bacterial organisms act like biological control agents with minimal impact on the environment. These microorganisms and their toxic metabolites should be included in those which are acceptable to qualify a product as organic or integrated and enjoy all the benefits that entails. Hence, interest in the use of microorganisms as biological agents to protect crop plants against plant parasitic nematodes has been increasing. This study investigated the ability of.Pseudomonas oryzihabitans symbiotically associated with the entomopathogenic nematode Steinernema abbasi as a bioagent against plant parasitic nematodes which infected tomato crop. The bacterium is particularly effective against root-knot nematode in vitro affecting the behavior and mobility of root-knot nematode juveniles. Also, studies in planta demonstrated the efficacy of P. oryzihabitans by preventing tomato root system by invasion of juveniles of Meloidogynejavaniea when bacterial cells were applied to the root system before nematodes. This efficacy is dependent on bacterial cell concentration used and the time of the nematode exposure. However, a better nematode control might be achieved with multiple applications of the biocontrol agent. Furthermore, the results of this study provide evidence that the bacterium P. oryzihabitans produces metabolites, which have nematostatic effects.

Molecular Characterization of Indian Species of Steinernema （Nematoda： Steinernematidae） Based on Restriction Fragment Length Polymorphism Profile of Internal Transcribed Spacer Region of Ribosomal DNA

Restriction fragment length polymorphism （RFLP） profiles of the amplified products of Internal Transcribed Spacer （ITS） region of rDNA using four restriction enzymes （Alul, Rsal, HinfI and HhaI） revealed distinctness of six Indian isolates of Steinernema one each from Maharashtra （IARI-EPN-mh）, Himachal Pradesh （IARI-EPN-hp）, Dehradun （IARI-EPN-dhdl）, Jharkhand （IARI-EPN-jhl） and two from Madhya Pradesh （IARI-EPN-bpll ＆ IARI-EPN-gwll）, when compared with the only native species Steinernema thermophilum. One of the restriction enzyme, Rsal could differentiate all the six species/strains from one another. The three restriction enzymes yielded patterns which were of diagnostic value but Rsal appeared to be the best diagnostic marker for differentiating these isolates. A tree constructed based upon the band sharing amongst the isolates, produced trichotomy which placed strains from Madhya Pradesh and Jharkhand in one group showing 94% homology, one strain from Bhopal （M.P） formed separate clade along with S. thermophilum with 72% similarity. These isolates, from Maharashtra, Himachal Pradesh and Dehradun, showed only 51% similarity with the S. thermophilum by forming separate clade.

Bemisia tabaci： The current situation in the UK and the prospect of developing strategies for eradication using entomopathogens

The sweetpotato whitefly, Bemisia tabaci （Gennadius） （Hemiptera： Aleyrodidae） remains a serious threat to crops worldwide. The damaging B-biotype is of specific economic concern because it is an effective vector of over 111 viruses from several fam- ilies, particularly geminiviruses. Bemisia tabaci is regularly intercepted on plants coming into the UK where it is subjected to a policy of eradication. The UK maintains Protective Zone status against this pest. A main pathway of entry of B. tabaci into the Protected Zone involves propagating material, especially Poinsettia （Euphorbia pulcherrima）. With increased insecticide resistance continuously being recorded, B. tabaci is becoming more difficult to control/eradicate. Recent research involving both entomopathogenic nematodes and fungi is showing much potential for the development of control programs for this pest. Both the nematode Steinernemafeltiae and the fungus Lecanicillium muscarium have been shown to be most effective against second instar B. tabaci. Fine-tuning of the environmental conditions required has also increased their efficacy. The entomopathogens have also shown a high level of compatibility with chemical insecticides, all increasing their potential to be incorporated into control strategies against B. tabaci.

Root symbionts： Powerful drivers of plant above- and belowground indirect defenses

Soil microbial mutualists of plants, including mycorrhizal ftmgi, non- mycorrhizal fungi and plant growth promoting rhizobacteria, have been typically characterized for increasing nutrient acquisition and plant growth. More recently, soil microbes have also been shown to increase direct plant defense against above- and below- ground herbivores. Plants, however, do not only rely on direct defenses when attacked, but they can also recruit pest antagonists such as predators and parasitoids, both above and belowground, mainly via the release of volatile organic compounds （i.e., indirect defenses）. In this review, we illustrate the main features and effects of soil microbial mutualists of plants on plant indirect defenses and discuss possible applications within the framework of sustainable crop protection against root- and shoot-feeding arthropod pests. We indicate the main knowledge gaps and the future challenges to be addressed in the study and application of these rnultifaceted interactions.