Neutral density liquid formulations for nematode-based biopesticides

Entomopathogenic nematodes are widely used as biological insecticides for agricultural and horticultural pests. In the vast majority of cases, commercial products contain nematodes partially dehydrated on to inert solid carriers. Nematode survival in these products is generally poor, they are difficult to handle and are not suitable for use with all nematode species. We have developed a non-viscous, non-adhesive and non-toxic liquid formulation for nematode storage and transport based on neutral density colloidal silica suspensions. Survival and virulence of nematodes stored in this formulation without aeration was superior to nematodes stored in aerated quarter strength Ringer's solution.     

Compatibility of Soil Amendments with Entomopathogenic Nematodes

The impact of inorganic and organic fertilizers on the infectivity, reproduction, and population dynamics of entomopathogenic nematodes was investigated. Prolonged (10- to 20-day) laboratory exposure to high inorganic fertilizer concentrations inhibited nematode infectivity and reproduction, whereas short (1-day) exposures increased infectivity. Heterorhabditis bacteriophora was more sensitive to adverse effects than were two species of Steinernema. In field studies, organic manure resulted in increased densities of a native population of Steinernema feltiae, whereas NPK fertilizer suppressed nematode densities regardless of manure applications. Inorganic fertilizers are likely to be compatible with nematodes in tank mixes and should not reduce the effectiveness of nematodes used for short-term control as biological insecticides, but may interfere with attempts to use nematodes as inoculative agents for long-term control. Organic manure used as fertilizer may encourage nematode establishment and recycling.     

Biological control potential of plant growth‐promoting rhizobacteria suppression of Meloidogyne incognita on cotton and Heterodera glycines on soybean: A review

Over the past decade, we have seen an increasing market for biopesticides and an increase in number of microbial control studies directed towards plant‐parasitic nematodes. This literature survey provides an overview of research on biological control of two economically important plant‐parasitic nematodes, Meloidogyne incognita (Kofoid & White) Chitwood (southern root‐knot nematode) and Heterodera glycines Ichinohe (soybean cyst nematode) using spore‐forming plant growth‐promoting rhizobacteria (PGPR). In this review, the current biological control strategies for the management of those cotton and soybean nematodes, the mechanism of using BacillusPGPR for biological control of plant‐parasitic nematode including induced systemic resistance and antagonism and the future of biological control agents on management of plant‐parasitic nematodes are covered.     

Control of Plutella xylostella using polymer-formulated Steinernema carpocapsae and Bacillus thuringiensis in cabbage fields

Abstract:  Field trials evaluating the potential of the entomopathogenic nematode Steinernema carpocapsae and the feasibility to combine nematodes with Bacillus thuringiensis for sustainable control of the diamondback moth (DBM) Plutella xylostella were conducted in cabbage cultivated in the province Probolinggo, east Java and Indonesia. A single use of 0.5 million S. carpocapsae  m⁻² applied with a surfactant-polymer-formulation containing 0.3% xanthan and 0.3% Rimulgan^® achieved a significant reduction of the insects per plant with >50% control after 7 days. Even 14 days after the application about 45% control was recorded and dead larvae containing nematodes were found. No significant effects were recorded when the formulation was compared with nematodes applied in water or with a surfactant alone. This was attributed to high humidity in the experimental area at the end of the rainy season and a microclimate in the cabbage heads favouring nematode survival. Weekly applications of B. thuringiensis (Turex^®) or alternating applications of Turex^® and the nematodes achieved >80% control. The application of both biological agents together every second week reached insignificant lower efficacy (70%). Nematodes can be used to substitute ineffective chemical insecticides and alterations with B. thuringiensis can prevent the further development of resistance against the bacterial control agent.     

Efficacy of soil treatments of entomopathogenic nematodes against the larvae, pupae and adults of Tuta absoluta and their interaction with the insecticides used against this insect

The efficacy of soil treatments of three native entomopathogenic nematodes (Steinernema carpocapsae, S. feltiae and Heterorhabditis bacteriophora) against Tuta absoluta larvae, pupae and adults was determined under laboratory conditions. The effect of three insecticides commonly used against T. absoluta, in the survival, infectivity and reproduction of these nematode strains was also evaluated. When dropped into soil to pupate, soil application of nematodes resulted in a high mortality of larvae: 100, 52.3 and 96.7 % efficacy for S. carpocapsae, S. feltiae and H. bacteriophora respectively. No mortality of pupae was observed and mortality of adults emerging from soil was 79.1 % for S. carpocapsae and 0.5 % for S. feltiae. The insecticides tested had a negligible effect on nematode survival, infectivity and reproduction. No sublethal effects were observed. Infective juveniles that survived to insecticide exposition were able to infect Galleria larvae with no significant differences from the control. The Galleria larvae affected by the three insecticides tested served as suitable hosts for the infection and reproduction of the nematodes. These results suggest that larvae of T. absoluta, falling from leaves following insecticide application, could be suitable hosts for nematodes, thereby increasing their concentration and persistence in the soil.     

Habitat associations of two entomopathogenic nematodes: a quantitative study using real-time quantitative polymerase chain reactions

1. Despite nematodes being the most abundant animals on earth, very few animal ecologists study them, probably because of the difficulties of identifying them to species by morphological methods. 2. A group of nematodes that are important both ecologically and economically is the entomopathogenic nematodes, which play a key role in regulating soil food webs and are sold throughout the world as biological insecticides, yet for which very little is known of their population ecology. 3. A novel detection and quantification method was developed for soil nematodes using real-time polymerase chain reaction (PCR), and the technique was used to estimate numbers of two closely related species of entomopathogenic nematodes, Steinernema kraussei and S. affine in 50 soil samples from 10 sites in Scotland representing two distinct habitats (woodland and grassland). 4. There was a high degree of correlation between our molecular and traditional morphological estimates of population size and our data clearly showed that Steinernema affine occurred only in grassland areas, whereas S. kraussei was found in grassland and woodland samples to a similar degree. 5. Real-time PCR offers a rapid and accurate method of detecting individual nematode species from soil samples without the need for a specialist taxonomist, and has much potential for use in studies of nematode population ecology.     

Effect of neonicotinoid synergists on entomopathogenic nematode fitness

In previous greenhouse and field studies, the neonicotinoid insecticide imidacloprid interacted synergistically with five entomopathogenic nematode species against five scarab species. Two other neonicotinoids, thiamethoxam and acetamiprid, showed a weaker interaction with nematodes in scarab larvae. Entomopathogenic nematodes have the potential to recycle in hosts after inundative applications, thereby increasing the persistence of nematodes and insect control. Thus we investigated the effect of neonicotinoids on nematode fitness after tank mixing and after combined applications. Tank mixing only had a negative effect on nematode survival and infectivity in a few nematode–insecticide combinations and only if both insecticide concentration and exposure time were several times higher than typical for field applications. Combined application of nematodes with imidacloprid generally had no negative effect on the percentage of scarab cadavers producing progeny or the number of nematode progeny emerging per cadaver. In experiments with a synergistic increase in scarab mortality, the total number of progeny in combination treatments was up to four times higher than in nematodes only treatments. Similarly, nematode populations in soil from combination treatments were 13.2 times greater than for nematodes only treatments at 28 days after treatment. Combined imidacloprid–nematode applications did not affect the pathogenicity or infectivity of the nematode progeny. Combining thiamethoxam with nematodes had no negative effects on nematode reproduction in the majority of treatments. However, due to the weaker interaction of thiamethoxam and nematodes on scarab mortality, the total number of nematode progeny per treatment generally did not increase compared with nematodes only treatments. The demonstrated tank mix compatibility of imidacloprid and nematodes improves the feasibility of combining these agents for curative white grub control. The positive effect of imidacloprid on nematode reproduction after combined application may increase the likelihood of infection of white grubs by subsequent generations of nematodes, thereby improving their field persistence and biological control potential.     

Colorado potato beetle control by application of the entomopathogenic nematode Heterorhabditis marelata and potato plant alkaloid manipulation

Control of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), with the entomopathogenic nematode Heterorhabditis marelata Liu and Berry (Nematoda: Heterorhabditidae) was examined in the laboratory and in potato fields in north central Oregon. This research tested the hypothesis that varying nitrogen fertilizer levels would affect foliar alkaloid levels, which would stress the host, and allow increased nematode reproduction and long‐term control of the CPB. Laboratory results indicated that nematodes tended to reproduce more readily in CPB fed on potato plants with high levels of fertilizer. Field trials tested CPB population responses to four treatments: application of nematodes vs. no nematodes, with application of low vs. high rates of nitrogen fertilizer. The higher nitrogen application rate increased field foliar levels of the alkaloids solanine by 35%, and chaconine by 41% over the season. Nematodes were applied twice during the season, causing a 50% reduction in adult CPB populations, and producing six times as many dead prepupae in nematode‐treated soil samples as in the untreated samples. However, no reproducing nematodes were found in the 303 dead prepupae and pupae collected from nematode‐treated plots. Nitrogen fertilizer levels, and their related alkaloid levels, did not affect nematode infection rates or reproduction in the field. Foliar alkaloid levels of plants from the growth chamber were 3–6‐fold as high as those in the field, which may explain the variation in nematode response to nitrogen applications to host plants of the CPB. Heterorhabditis marelata is effective for controlling CPB in the field, and does not have negative non‐target effects on one of the most common endemic CPB control agents, Myiopharus doryphorae (Riley) (Diptera: Tachinidae), but the low rate of nematode reproduction cannot be manipulated through alkaloid stress to the beetle. Until H. marelata can be mass‐produced in an inexpensive manner, it will not be a commercially viable control for CPB.     

Quantification of the slug parasitic nematode Phasmarhabditis hermaphrodita from soil samples using real time qPCR

Phasmarhabditis hermaphrodita is a nematode parasite that infects and kills several species of slugs. The nematode is produced commercially as a biological control agent for slug pests of agriculture and horticulture. Given the difficulties of distinguishing this species from other nematode species in soil samples, very little is known about its natural ecology or its behaviour and persistence following application for biological control. Here we describe a method to quantify P. hermaphrodita in soil samples based on real time PCR. We designed primers and a dual labelled fluorescent probe that can be used to quantify numbers of P. hermaphrodita and which is capable of distinguishing this species from the morphologically identical Phasmarhabditis neopapillosa. We compared different methods whereby the entire nematode community is extracted prior to DNA extraction, and three methods to extract DNA directly from soil samples. Both nematode extraction and DNA extraction from large (10 g) samples of soil gave reliable estimates of nematode numbers, but methods which extracted DNA from small (1 g or less) soil samples substantially underestimated numbers. However, direct extraction of DNA from soils may overestimate numbers of live nematodes as DNA from dead nematodes was found to persist in soil for at least 6 days. The technique could be modified for detection and quantification of all soil borne parasitic nematodes.     

Evaluation of hydraulic,pneumatic and mechanical agitation for the spray application of Steinernema carpocapsae (Rhabditida: Steinernematidae)

The application of entomopathogenic nematodes (EPN) is generally done using standard spray application techniques. However, in contrast to chemical pesticides, these biological antagonists must remain viable during and after the application process. For the application of EPN, a good agitation system is indispensable as the nematodes tend to sediment fast in a spray tank without agitation. Three agitation systems, viz. mechanical, pneumatic and hydraulic agitation were tested for their ability to keep Steinernema carpocapsae (Rhabditida: Steinernematidae) suspended in an undamaged way. Hydraulic agitation was tested using a centrifugal and a diaphragm pump. Nematode damage was quantified based on viability and infectivity of the EPN. The ability of the agitation system to keep the nematodes in suspension was examined by comparing the nematode concentration observed in the samples taken at different agitation times. Only the hydraulic agitation using the centrifugal pump damaged the nematodes. After 120 min of recirculation, only 19.3% of the nematodes survived. Infectivity was even reduced to 0%. An additional experiment revealed that the temperature rise, from 21.7 to 45.4°C, was responsible for the observed nematode damage. The concentration measurements showed that the pneumatic agitation was unstable. Agitation during 120 min using the other agitation systems resulted in a significant loss of nematodes at 15 cm above the spray tank bottom. In conclusion, mechanical and hydraulic agitation using a diaphragm pump can be recommended when S. carpocapsae is applied, although attention should be paid to possible nematode loss during application.     

Comparative effects of Steinernema feltiae (Nematoda: Steinernematidae) and insecticides on yield and cropping of the mushroom Agaricus bisporus

In a mushroom crop (Agaricus bisporus) affected by a very low level of sciarid fly (Lycoriella auripila) infestation, the effects of an indigenous isolate of insect-parasitic nematode (Steinernema feltiae) and of two commonly used insecticides (diazinon and diflubenzuron) were studied. When compared with untreated plots, nematodes applied to the casing had no adverse effects on mushroom yields whereas insecticides decreased yields. At a rate of 3 × 10⁶ infective juveniles per tray (surface area = 0.56 m²), S. feltiae elicited increases of 28.5% and 19% in the mean total numbers and weights of mushrooms respectively. Treatment only with diflubenzuron resulted in 14.6% and 6% reductions in mean total numbers and weights of mushrooms, respectively; treatment with both diazinon and diflubenzuron caused 18.5% and 9.4% losses. Application of nematodes generally reduced the mean weight per mushroom whereas insecticides increased it; nematodes delayed the onset of mushroom production (first flush) whereas diflubenzuron delayed the third and fourth flushes. Nematode contamination of sporophores was minimal when S. feltiae was applied at casing. Although their numbers declined with time, the nematodes persisted, in the casing layer, throughout the cropping period of seven weeks. It is concluded that yield benefits associated with nematode application can result mainly from nematode effects on A. bisporus and not solely from suppression of a damaging pest population.     

Application and commercialization of nematodes

While nematodes are most commonly known for their negative impact on plants, animals, and humans, there are a number of species which are commercially explored. This review highlights some of the most important success stories for the application of nematodes. They are used as bioindicators in ecological and toxicity studies, as model organisms for elucidating fundamental biological questions and for high throughput screening of drugs. Besides these indirect uses, direct applications include the use of Beddingia siricidicola against a major forest pest and the commercialization of Steinernema, Heterorhabditis, and Phasmarhabditis as biological pest control products. New directions for the commercialization of nematodes are the use as living food, specifically loaded with essential nutrients for various fish and shrimp larvae. Even human parasites or closely related species have been successfully used for curing autoimmune disorders and are currently in the process of being developed as drugs. With the striving development of life sciences, we are likely to see more applications for nematodes in the future. A prerequisite is that we continue to explore the vast number of yet undiscovered nematode species.     

Pineapple Nematode Research in Hawaii: Past,Present, and Future

The first written record of pineapple in Hawaii is from 1813. In 1901 commercial pineapple production started, and in 1924 the Experiment Station for pineapple research was established. Nematode-related problems were recognized in the early 1900s by N. A. Cobb. From 1920 to approximately 1945 nematode management in Hawaiian pineapple was based on fallowing and crop rotation. During the 1920s and 1930s G. H. Godfrey conducted research on pineapple nematode management. In the 1930s and 1940s M. B. Linford researched biological control and described several new species of nematodes including Rotylenchulus reniformis. In 1941 nematology and nematode management were advanced by Walter Carter''s discovery of the first economical soil fumigant for nematodes, D-D mixture. Subsequently, DBCP was discovered and developed at the Pineapple Research Institute (PRI). Since 1945 soil fumigation has been the main nematode management strategy in Hawaiian pineapple production. Recent research has focused on the development of the nonvolatile nematicides, their potential as systemic nematicides, and their application via drip irrigation. Current and future research addresses biological and cultural alternatives to nematicide-based nematode management.     

The bacterial community of entomophilic nematodes and host beetles

Insects form the most species‐rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode–insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle–nematode–bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five‐year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high‐throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate‐reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect‐associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate‐reducing bacteria suggests a possible link between beetle–bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.     

Ecology in the service of biological control: the case of entomopathogenic nematodes

 Biological control manipulations of natural enemies to reduce pest populations represent large-scale ecological experiments that have both benefited from and contributed to various areas of modern ecology. Unfortunately, economic expediency and the need for rapid implementation often require that biological control programs be based more on trial and error than on sound ecological theory and testing. This approach has led to some remarkable successes but it has also produced dismal failures. This point is particularly well illustrated in the historical development and use of entomopathogenic nematodes for the biological control of insect pests. Intense effort has focused on developing these natural enemies as alternatives to chemical insecticides, in part because laboratory assays indicated that these nematodes possess a broad host range. This illusory attribute launched hundreds of field releases, many of which failed due to ecological barriers to infection that are not apparent from laboratory exposures, where conditions are optimal and host-parasite contact assured. For example, the entomopathogenic nematode Steinernema carpocapsae is a poor choice to control scarab larvae because this nematode uses an ambusher foraging strategy near the soil surface whereas the equally sedentary scarab remains within the soil profile, shows a weak host recognition response to scarabs, has difficulty overcoming the scarab immune response, and has low reproduction in this host. Conversely, two other nematodes, Heterorhabditis bacteriophora and S. glaseri, are highly adapted to parasitize scarabs: they use a cruising foraging strategy, respond strongly to scarabs, easily overcome the immune response, and reproduce well in these hosts. Increased understanding of the ecology of entomopathogenic nematodes has enabled better matches between parasites and hosts, and more accurate predictions of field performance. These results underline the importance of a strong partnership between basic and applied ecology in the area of biological control. Received: 15 July 1996 / Accepted: 5 November 1996     

Parameters affecting plant defense pathway mediated recruitment of entomopathogenic nematodes

Entomopathogenic nematodes are natural enemies and effective biological control agents of subterranean insect herbivores. Interactions between herbivores, plants, and entomopathogenic nematodes are mediated by plant defense pathways. These pathways can induce release of volatiles and recruit entomopathogenic nematodes. Stimulation of these plant defense pathways for induced defense against belowground herbivory may enhance biological control in the field. Knowledge of the factors affecting entomopathogenic nematode behaviour belowground is needed to effectively implement such strategies. To that end, we explore the effect of elicitor, elicitor dose, mechanical damage, and entomopathogenic nematode release distance on recruitment of entomopathogenic nematode infective juveniles to corn seedlings. Increasing doses of methyl jasmonate and methyl salicylate elicitors recruited more entomopathogenic nematodes as did mechanical damage. Recruitment of entomopathogenic nematodes was higher at greater release distances. These results suggest entomopathogenic nematodes are highly tuned to plant status and present a strategy for enhancing biological control using elicitor-stimulated recruitment of entomopathogenic nematodes.     

A ribosomal DNA-based framework for the detection and quantification of stress-sensitive nematode families in terrestrial habitats

Indigenous communities of soil‐resident nematodes have a high potential for soil health assessment as nematodes are diverse, abundant, trophically heterogeneous and easily extractable from soil. The conserved morphology of nematodes is the main operational reason for their under‐exploitation as soil health indicators, and a user‐friendly biosensor system should preferably be based on nonmorphological traits. More than 80% of the most environmental stress‐sensitive nematode families belong to the orders Mononchida and Dorylaimida. The phylogenetic resolution offered by full‐length small subunit ribosomal DNA (SSU rDNA) sequences within these two orders is highly different. Notwithstanding several discrepancies between morphology and SSU rDNA‐based systematics, Mononchida families (indicated here as M1–M5) are relatively well‐supported and, consequently, family‐specific DNA sequences signatures could be defined. Apart from Nygolaimidae and Longidoridae, the resolution among Dorylaimida families was poor. Therefore, a part of the more variable large subunit rDNA (≈ 1000 bp from the 5′‐end) was sequenced for 72 Dorylaimida species. Sequence analysis revealed a subclade division among Dorylaimida (here defined as D1–D9, PP1–PP3) that shows only distant similarity with ‘classical’ Dorylaimid systematics. Most subclades were trophically homogeneous, and — in most cases — specific morphological characteristics could be pinpointed that support the proposed division. To illustrate the practicability of the proposed molecular framework, we designed primers for the detection of individual subclades within the order Mononchida in a complex DNA background (viz. in terrestrial or freshwater nematode communities) and tested them in quantitative assays (real‐time polymerase chain reaction). Our results constitute proof‐of‐principle for the concept of DNA sequence signatures‐based monitoring of stress sensitive nematode families in environmental samples.     

Field Evaluation of Steinernema feltiae Against the Web-spinning Larch Sawfly Cephalcia lariciphila

Field trials were conducted in Rheola Forest, Wales, Great Britain, to determine the effectiveness of Steinernema feltiae UK strain in controlling the web-spinning larch sawfly Cephalcia lariciphila. Foliar sprays at the rate of 5,000-20,000 nematodes/100 cm branch resulted in 3.4-29.4% infection of sawfly larvae. Soil application of 200 nematodes/cm² resulted in 61% infection of sawfly prepupae and 17.3% of pupae. Prepupal infection ranged from 4.8 to 14.7% 1 year after nematode application. Soil applications of this nematode show that it has potential for biological control of sawfly prepupae.     

Insect Biocontrol with Non-endemic Entomopathogenic Nematodes (Steinernema and Heterorhabditis spp.): Conclusions and Recommendations of a Combined OECD and COST Workshop on Scientific and Regulatory Policy Issues

Fifteen invited experts from 10 Organisation for Economic Cooperation and Development (OECD) and European countries participating in the European Commission's Cooperation in the Field of Science and Technical Research (COST) Action 819, along with 12 other participants, met to review and debate the potential problems associated with the introduction and commercial use of non-indigenous nematodes for insect biological control. The consensus view of the participants was that entomopathogenic nematodes (EPNs) possess specific biological and ecological features, which make their use in biological control exceptionally safe. All the scientific evidence available supports the conclusion that EPNs are safe to the environment, as well as to production and application personnel, the general public and the consumers of agricultural products treated with them. Only a few potential, but very remote, risks could be identified. Therefore, it was recommended that EPNs should not be subject to any kind of registration. The introduction of non-indigenous nematode species, however, should be regulated. Species should be accurately identified, and details of the origin, known distribution, probable host range and safety to the user must be provided. In addition, an expert opinion, based on available information, of the possible impact on non-target organisms must be available.