Post-application Persistence of Entomopathogenic Nematodes

Five phases can be distinguished in the post-application persistence of entomopathogenic nematodes and each phase is associated with a specific set of mortality factors. Pre-application factors associated with production, storage and transport conditions determine the survival rate and quality of nematodes at the time of application. The phase of tank mixing and application with a sprayer, hose or other equipment does not usually cause mortality as nematode dauer juveniles are quite tolerant of shear forces. The most critical periods for survival are the first few minutes and hours directly after application. High losses, in the order of 40-80%, often occur during this phase. Ultraviolet radiation and dehydration are probably the most important mortality factors. The remaining nematodes settle in the soil and their numbers gradually decrease at levels of 5-10% per day. Predation, infection by antagonists, depletion of energy and desiccation are probably the main mortality factors during this period. In most cases, after 2-6 weeks less than 1% of the applied population is still alive. Through recycling in host insects, nematodes may then persist for years at these levels. Thus, the pattern is a rapid decline in the first few days followed by a moderate decline over the next 2-6 weeks and then a long period of recycling at a low level. Some nematode species that normally occur in warmer climatic zones can also persist in colder climates. Major side-effects of applications of entomopathogenic nematodes are not likely to occur as the population density decreases to background levels within days or weeks after application. Furthermore, there is little or no migration of the nematodes to neighbouring fields. The relatively short period of persistence of entomopathogenic nematodes and the necessity of their populations to recycle frequently in hosts in order to survive make it unlikely that they could have major effects on non-target organisms. Their selectivity and beneficial traits as biological control agents outweigh the small risks of causing unwanted environmental disturbance in non-target populations.     

Survival Mechanisms of Entomopathogenic Nematodes

Entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) are isolated from a wide variety of ecosystems ranging from sub-Arctic to arid and tropical climates. It is expected that natural populations of these nematodes will be pre-adapted to specific ecological conditions in their environments. However, little is known about the survival strategies of entomopathogenic nematodes. This review focuses on the main survival mechanisms involved in the long-term persistence of nematodes: extreme temperature tolerance (cold and heat), desiccation tolerance, osmotic tolerance, pesticide resistance and limitations of aeration. Physiological and biochemical mechanisms for survival are discussed. The review addresses the implications of the survival strategies on the establishment of exotic and indigenous steinernematids and heterorhabditids.     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

Yersinia pseudotuberculosis nucleoside-kinase

Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.     

Yersinia pseudotuberculosis toxins

The review of publications about protein toxins Y. pseudotuberculosis are presented. It includes the main data obtained by domestic and foreign investigators as well as the results of our own elaboration in the study of Y. pseudotuberculosis protein toxins. The guestions of isolation, purification, characterization of physico-chemical and biological properties, the mechanism action and role of toxins on pathogenesis of infection were discussed.     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

Efficacy and Persistence of Entomopathogenic Nematodes in Controlling Larval Populations of Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae)

A 3-year study was conducted in a Pinus halepensis reforestation of Apulia Region (Southern Italy) injecting IJs (infective juveniles) of Steinernema feltiae , S. carpocapsae and Heterorhabditis bacteriophora in aqueous and gel suspensions (Idrosorb SR 2002 [Nigem ® ], and Compex) into the nests of Thaumetopoea pityocampa caterpillar. This study showed that the gel suspensions do not percolate and that slow release of water from the gels allowed nematodes to survive and complete their life cycle in the host. Results demonstrate the feasibility of reducing overwintering larval populations by injecting gel suspension of S. feltiae . We found no negative effects on the endoparasite Phryxe caudata .     

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.     

Quality Assessment of Commercially Produced Entomopathogenic Nematodes

The quality of the most widely commercialized entomopathogenic nematodes, Steinernema carpocapsae (Weiser) and Heterorhabditis bacteriophora Poinar, was assessed from 30 shipments from six United States suppliers mailed to three locations. These suppliers comprise a cottage industry aimed in large part at a mail-order market. Most companies were accessible and reliably shipped pure populations of the correct species on time, in sturdy containers, and often with superb accompanying instructions. Nematodes were received in satisfactory condition with acceptable levels of viability. Consistency, however, was a problem, with each supplier having one or more weak spots to bolster. Most shipments did not contain the expected nematode quantity, and one shipment had no nematodes. Pathogenicity of several products against Galleria mellonella (L.) larvae was not equivalent to our controls. H. bacteriophora was not always available when ordered. A few products contained mixed populations of both nematode species. Application rate recommendations provided by some suppliers appeared unsound. We conclude that (1) the entomopathogenic nematode cottage industry lacks rigorous quality control, (2) self-regulation is problematic without feedback on quality, and (3) consumers are rarely able to provide this feedback. Improved reliability by the nematode industry will most likely be achieved via industry-generated agreement on standards for quality.     

Evolution of Host Search Strategies in Entomopathogenic Nematodes

There is interspecific variation in infective juvenile behavior within the entomopathogenic nematode genus Steinernema. This variation is consistent with use of different foraging strategies along a continuum between ambush and cruise foraging. To address questions about the evolution of foraging strategy, behavioral and morphological characters were mapped onto a phylogeny of Steinernema. Three species, all in the same clade, were classified as ambushers based on standing bout duration and host-finding ability. One clade of six species were all cruisers based on both host-finding and lack of standing behavior. All species in the ambusher clade had a high rate of jumping, all species in the cruiser clade had no jumping, and most intermediate foragers exhibited some level of jumping. Response to volatile and contact host cues was variable, even within a foraging strategy. Infective juveniles in the ambusher clade were all in the smallest size category, species in the cruiser clade were in the largest size categories, and intermediate foragers tended to be more intermediate in size. We hypothesize that the ancestral Steinernema species was an intermediate forager and that ambush and cruise foraging both evolved at least once in the genus.     

Genetic Enhancement of Nematicide Resistance in Entomopathogenic Nematodes

Entomopathogenic nematodes are highly effective bioinsecticides . Their efficacy may be reduced due to the various pesticides they encounter in the soil . These include insecticides as well as nematicides used against plant - parasitic nematodes . The purpose of this study was to examine the feasibility of genetic selection as a means of enhancing resistance of the entomopathogenic nematode Heterorhabditis bacteriophora strain HP88 to the nematicides: Fenamiphos (an organophosphate) , Oxamyl (a carbamate) and Avermectin (a biological product) . Estimates of heritability ( h 2) of resistance to the three nematicides were obtained from analysis of inbred lines derived from the base population . The heritability estimate for Fenamiphos was h 2 = 0 . 31 , for Oxamyl h 2 = 0 . 71 and for Avermectin h 2 = 0 . 46 . Five rounds of selection were performed . Thereafter , each line was divided into two: for one subline selection continued for six additional rounds . The other subline was reared without selection for the six additional rounds . After the eleventh round , resistance to the nematicides was examined as were several traits relevant to biocontrol efficacy including virulence , heat tolerance and reproduction potential . Selection resulted in an 8 - 9 - fold increase in resistance to Fenamiphos and Avermectin and a 70 - fold increase in resistance to Oxamyl . The enhanced resistance Oxamyl and Avermectin , and to a lesser extent to Fenamiphos , was stable and continued after selection was relaxed . No deterioration in traits relevant to biocontrol efficacy was observed in the selected lines as compared with the base population . The selected lines displayed enhanced cross - resistance towards some , but not all , of the nematicides tested . These results demonstrate that genetic selection can be used to enhance resistance of entomopatho genic nematodes to certain environmental stresses . The selected lines will be useful bioinsecti cides in the context of integrated pest management .     

Pathogenicity and Host Specificity of Entomopathogenic Nematodes

The mechanisms of infection and pathogenicity of Steinernematidae and Heterorhabditidae in insect hosts are discussed as factors influencing the host specificity of these nematodes. The invasion and evasion of host defences are important steps in the pathogenic process. The ability of the nematode to penetrate into the insect haemocoel, achieved by the release of proteolytic enzymes, is one specific factor. Another specific factor in the nematode-insect relationship is the ability of the nematode to evade insect defences through failure to be recognized and/or by destruction of insect antibacterial factors. Toxins and extracellular enzymes are important virulence factors released by these nematodes, apparently exhibiting a specific activity against certain insect hosts.     

Impact of Entomopathogenic Nematodes on Non-target Hosts

Biological pest control has been thought to be ecologically safe for many years. More recently, it has been questioned whether entomopathogens and beneficial arthropods or nematodes truly have no impact on non-target species. Only a few studies deal with the action of entomopathogenic nematodes on non-target animals, although a broad spectrum of species has been tested in the laboratory. Entomopathogenic nematodes do not affect vertebrates under natural conditions. Mortality caused by the release of entomopathogenic nematodes among non-target arthropod populations can occur, but will only be temporary, will be spatially restricted and will affect only part of a population. In plots treated with entomopathogenic nematodes, the impact on the non-target fauna proved to be negligible. The possible impact of introduced exotic nematode species is discussed and regulatory measures for the release are proposed.     

Community outbreak of Yersinia pseudotuberculosis

An outbreak of Yersinia pseudotuberculosis in Kurashiki, Japan is described. This is the first conclusive report of a community outbreak of this microorganism. A total of 535 pupils, five teachers, and one food attendant contracted the organism. Causative organisms were detected in 19 out of 30 patients. All isolated strains belonged to serotype VA. Out of 653 sera of the pupils, 488 showed elevated agglutinin titers ranging from 1:80 to 1:1,280 or more within a period of 3 months.     

Postdiarrheal arthropathy of Yersinia pseudotuberculosis.

Two patients with acute gastroenteritis in whom polyarthritis subsequently developed were found to have positive serologic results for Yersinia pseudotuberculosis. With resolution of the arthropathy the antibody titres decreased. While the patient without the histocompatibility antigen HLA-B27 had an acute, self-limited arthritis, the patient with this antigen had a more chronic arthritis. Serologic typing and stool culture for Y. pseudotuberculosis should be done in cases of postdysenteric arthritis.     

Action of sodium chloride on Yersinia pseudotuberculosis and Yersinia enterocolitica

The bacteriostatic and bactericidal action of sodium chloride on 60 Y. pseudotuberculosis strains, 75 Y. enterocolitica strains and 158 urine-fermenting strains has been studied. A new specific feature of Y. pseudotuberculosis has been revealed: high sensitivity to sodium chloride. The suitability of the sodium chloride test has been shown for the identification of Yersinia and the differentiation of Y. pseudotuberculosis and Y. enterocolitica.     

Effect of acidin-pepsin on Yersinia pseudotuberculosis

The effect of acidin-pepsin solution at a concentration of 1 : 100 on newly isolated Yersinia cultures has been tested in three series of experiments. Acidin-pepsin has been found to exert a bactericidal effect on Yersinia and to induce the appearance of involutionary forms and large rod-shaped Yersinia cells with a better capacity for survival. The surviving Yersinia cells do not pass their resistance to acidin-pepsin on to their progeny and show low invasiveness and pathogenicity in white mice.