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Deroceras laeve Müller and D. reticulatum Müller are serious slugs of field crops in Egypt. Chemical control using metaldehyde baits is still the main control tactic. In 2018, a survey of terrestrial slugs at Belbis district, Sharkia Governorate, Egypt failed to find the molluscicidal nematode, Phasmarhabditis hermaphrodita. Dead slugs produced only free-living or non pathogenic nematodes belong to Family Rhabditidae. Two imported EPNs species; Steinernema carpocapsae (All strain) and Heterorhabditis bacteriophora (HP88 strain) were screened for molluscicidal activity against D. laeve and D. reticulatum compared with three Egyptian Heterorhabditis isolates, Ht strain, Ar-4 strain and Serag1 strain. S. carpocapsae (All strain) and H. bacteriophora (HP88 strain), showed promising results by killing territorial slugs. S. carpocapsae (All strain) resulted in the greatest percentage mortalities 100% in D. laeve and D.reticulatum after 14 days at a concentration of 2000IJs/cm whereas, the application of H. bacteriophora (HP88 strain) induced percentages mortality 50 and 66.67% with D. laeve and D. reticulatum, respectively. The Egyptian strain H. bacteriophora showed less molluscicidal activity against the two tested slugs. After 14 days, D. reticulatum mortalities were 36.67, 40.00 and 46.33 % by the application of H. bacteriophora (Serag1 strain), H. bacteriophora (Ht strain) and H. bacteriophora (Ar-4 strain), respectively. Whereas, the percentage mortalities were 20.00, 23.33 and 26.67 % ; 10.00, 13.33 and 13.33% in D. leave at low concentrations (1000 IJs/cm 2 & 500 IJs/cm 2), respectively. Egyptian isolates showed less encourage usage as a specific biological control agent against D.laeve and D.reticulatum compared to S. carpocapsae (All strain) and H. bacteriophora (HP88 strain). Finally, results of this study indicate the need to an extensive survey in all Egypt to detect and isolate the slug parasitic nematode, P. hermaphrodita as it was recorded in Dakahlia governorate infesting different snails and slug species and research should proceed to find virulent isolates from EPNs to control terrestrial slugs.
Controlling aflatoxigenic Aspergillus flavus and aflatoxins (AFs) in grains and food during storage is a great challenge to humans worldwide. Alcaligenes faecalis N1-4 isolated from tea rhizosphere soil can produce abundant antifungal volatiles, and greatly inhibited the growth of A. flavus in un-contacted face-to-face dual culture testing. Gas chromatography tandem mass spectrometry revealed that dimethyl disulfide (DMDS) and methyl isovalerate (MI) were two abundant compounds in the volatile profiles of N1-4. DMDS was found to have the highest relative abundance (69.90%, to the total peak area) in N1-4, which prevented the conidia germination and mycelial growth of A. flavus at 50 and 100 μL/L, respectively. The effective concentration for MI against A. flavus is 200 μL/L. Additionally, Real-time quantitative PCR analysis proved that the expression of 12 important genes in aflatoxin biosynthesis pathway was reduced by these volatiles, and eight genes were down regulated by 4.39 to 32.25-folds compared to control treatment with significant differences. And the A. flavus infection and AFs contamination in groundnut, maize, rice and soybean of high water activity were completely inhibited by volatiles from N1-4 in storage. Scanning electron microscope further proved that A. flavus conidia inoculated on peanuts surface were severely damaged by volatiles from N1-4. Furthermore, strain N1-4 showed broad and antifungal activity to other six important plant pathogens including Fusarium graminearum, F. equiseti, Alternaria alternata, Botrytis cinerea, Aspergillus niger, and Colletotrichum graminicola. Thus, A. faecalis N1-4 and volatile DMDS and MI may have potential to be used as biocontrol agents to control A. flavus and AFs during storage.
Parasites and parasitic lifestyles have evolved from free-living organisms multiple times. How such a key evolutionary transition occurred remains puzzling. Facultative parasites represent potential transitional states between free-living and fully parasitic lifestyles because they can be either free-living or parasitic depending on environmental conditions. We suggest that facultative parasites with phenotypically plastic life-history strategies may serve as evolutionary stepping-stones towards obligate parasitism. Pre-adaptations provide a starting point for the transition towards opportunistic or facultative parasitism, but what evolutionary mechanism underlies the transition from facultative to obligate parasitism? In this Opinion Piece, we outline how facultative parasites could evolve towards obligate parasites via genetic assimilation, either alone or in combination with the Baldwin effect. We further describe the key predictions stemming from each of these evolutionary pathways. The importance of genetic assimilation in evolution has been hotly debated. Studies on facultative parasites may not only provide key insights regarding the evolution of parasitism, but also provide ideal systems in which to test evolutionary theory on genetic accommodation.
Entomopathogenic nematodes from the two genera Steinernema and Heterorhabditis are widely used as biological agents against various insect pests and represent a promising alternative to replace pesticides. Efficacy and biocontrol success can be enhanced through improved understanding of their biology and ecology. Many endogenous and environmental factors influence the survival of nematodes following application, as well as their transmission success to the target species. The aim of this paper is to give an overview of the major topics currently considered to affect transmission success of these biological control agents, including interactions with insects, plants and other members of the soil biota including conspecifics.
Schistosomiasis is a parasitic disease transmitted to man and different warm blooded animals by means of snails. Great effort has been made to control the transmission of the disease by many strategies. Consequently, the utilization of particular molluscicides is viewed as a standout amongst the best measures for molluscs control. Recently, microbial pathogen used as non-traditional molluscicides which have attracted significant research attention due to the increasing, worldwide development of resistance to chemical molluscicides in molluscs populations. The present work aimed to study the molluscicidal impacts of eleven isolates of Bacillus thuringiensis which were isolated from soils of six Egyptian governorates toward Biomphalaria alexandrina snails. Results showed that, B. thuringiensis provides an effective biological control agent against B. alexandrina snails. Out of the tested isolates, four isolates; Qalyubia, Asyut 1, Qena and North Sinai 2 isolates show high-level molluscicidal activity. The obtained results indicated that LC50 and LC90 values were ranged between 133.27 - 457.74 mg/mL and 270.32 - 781.05 mg/mL, respectively. The most noteworthy molluscicidal impact was displayed by Qalyubia isolate which isolated from Qalyubia governorate with mortality rate extended from 20-100% at five treatment concentrations of 100-500 mg/mL. The LC50 and LC90 values for Qalyubia isolate were 133.27 mg/mL and 270.32 mg/mL, respectively.
Nematodes are important, largely because they frequently act as parasites and threaten the health of plants, animals and even humans. Here, we describe an interesting free-living nematode from land snails on Luofu Mountain, Guangdong, China. Alloionema luofuensis, sp. nov. is phylogenetically related to slug-parasite A. appendiculatum and the well-known vertebrate parasites Strongyloides spp. based on small subunit (SSU) and the D2-D3 domain of large subunit (LSU) rDNA sequences. The new species possesses an extremely transparent body and is easily maintained using C. elegans culture media, suggesting a possible application prospect of this free-living nematode as a comparative model system for its related parasites. Morphology and anatomy of the gonochoristic A. luofuensis, sp. nov. adult were described and illustrated. The species is characterised by a filiform tail bisexually, ‘rhabditiform’ oesophagus and ‘rhabditid-like’ female anatomy, but its male caudal region is completely different from that of typical rhabditid nematodes, being absent from an enveloping bursa. It is the first marker taxon characterised morphologically as well as molecularly from the family Alloionematidae, a group of nematodes with hyperdiverse molecular genetic variations underlying highly conserved anatomy. Further molecular and genetic studies on A. luofuensis, sp. nov. populations hold promise to provide insight into evolution of the clade consisting of vertebrate parasites of the heterogonic nematode genus Strongyloides. This is because of its unusual high levels of heterozygosity maintained by the conserved rRNA genes of partial SSU and the D2-D3 domain of LSU for the type isolate of this species.
Bacterial strains associated with entomopathogenic nematodes (EPNs) Rhabditis (Oscheius) spp. were isolated from infected cadavers of Galleria mellonella. The obtained 18 isolates were subdivided into nine phylogenetically different genera based on comparative sequence analysis of their 16S rRNA genes. The isolates were affiliated to three different class namely γ-proteobacteria (Enterobacter, Proteus, Providencia, Pseudomonas, Stenotrophomonas), β-proteobacteria (Alcaligenes) and Bacilli (Bacillus, Enterococcus, Lysinibacillus). It was observed that Gram-positive strains (Bacilli) were more frequently associated with the EPN, whereas Gram-negative isolates were affiliated to six different genera with more genotypic diversity. Subsequently, all bacterial isolates used in this study were analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Eight restriction endonucleases (CfoI, HinfI, RsaI, DdeI, Sau3AI, AluI, HaeIII, and MspI) were examined and a total of 15 different genotypes were obtained, forming two heterogenous main clusters after analysis by un-weighted pair-group method using arithmetic averages.
Arion vulgaris is one of the most important slug pests of crop plants, and is extremely hard to control. We assessed the effectiveness of a slug pellets containing iron phosphate, compared with methiocarb-and metaldehyde-based molluscicides, and of the parasitic nematode P. hermaphrodita contained in the commercial bioproduct Nemaslug. The effect of various doses of these substances and the nematode in reducing slug damage to Chinese cabbage plants was evaluated in laboratory experiments. A reduction in slug numbers and plant damage was obtained following application of the molluscicides, and a reduction in plant damage following application of P. hermaphrodita (immersion of plant roots in the nematode-containing liquid). The results are of great practical significance, because they show that it is possible to reduce the recommended dose of iron phosphate (5 g/m 2) by one half. Another important finding is that application of the nematode in the form of root immersion is more effective than spraying. The study demonstrates the usefulness of the nematode and of the iron phosphate molluscicide in protecting plants from A. vulgaris; this is of particular importance for crops on which the application of traditional molluscicides is not possible.
A survey of nematodes associated with terrestrial slugs was conducted for the first time in Norway. A total of 611 terrestrial slugs were collected from 32 sample sites. Slugs were identified by means of morphological examination, dissection of genitalia and molecular analysis using mitochondrial DNA. Twelve slug species were identified, representing four different slug families. Internal nematodes were identified by means of morphological analysis and the sequencing of the 18S rRNA gene. Of the sample sites studied, 62.5% were found to be positive for nematode parasites, with 18.7% of all slugs discovered being infected. Five nematode species were identified in this study: Alloionema appendiculatum, Agfa flexilis, Angiostoma limacis, Angiostoma sp. and Phasmar-habditis hermaphrodita. Of these species, only one nematode was previously undescribed (Angiostoma sp.). This is the first record of the presence of A. appendiculatum, A. flexilis and A. limacis in Norway.
Pest snails are economically important pests of the grain industry. Nematode based bioagent appears to be a hope for controlling pest snails in an environment friendly way. Based on the dataset of 18S rRNA gene sequences, we propose a molecular phylogeny of nematodes baited with Cernuella virgata in soils collected from southern states of Australia. A total of 12 species (representing eight genera of nematodes) were identified and the inferred phylogenetic trees (Neighbor-Joining and Minium Evolution) placed them within three (I, IV and VII) out of the seven clades, indicating the possibility of multiple origins of snail parasitism. In Clade I and Clade VII, nematodes associated with Cernuella virgata formed sister group relationships with some slug – parasitic nematodes. We assume that snail – parasitic nematodes and slug-parasitic nematodes might share common ancestors in their evolutionary histories.
EL-Beheira governorates in northwestern Egypt. Five
land snail species at Abees region on grape orchard
including Eobania vermiculata, Theba pisana, Helicella
vestalis, Monacha obstructa and Oxychillus alliarius; while
two species at EL-Mamoura region on ornamental plants
including E. vermiculata and T. pisana were recorded in
Alexandria governorate. In EL-Beheria governorate,
three land snail species including T. pisana, H. vestalis and
M. obstructa were recorded on navel orange and apple
trees at Kafr EL-Dwar center and E. vermiculata, T.
pisana and C. acuta were found on ornamental plants at
Abulmatamir center. The most abundant species were T.
pisana and E. vermiculata at Alexandria (71.4 and 25.5%)
and El-Beheira (65.6 and 14.2%) respectively, while the
lowest found were O. alliarius (0.5%) at Alexandria and
M. obstructa (4.1%) at EL-Beheira.
A polyphasic taxonomic study was performed on nine isolates recovered from various human clinical samples. Phenotypically, these isolates resembled Alcaligenes faecalis. Whole-cell protein analysis distinguished two different species, and this was confirmed by DNA-DNA hybridizations. Cellular fatty acid analysis and 16S rDNA sequence analysis indicated that these isolates were related to the genera Alcaligenes, Bordetella, Achromobacter and Pigmentiphaga and belonged to the family Alcaligenaceae. On the basis of the results of this study, the organisms were classified in a novel genus, Kerstersia gen. nov. This genus comprises one species, Kerstersia gyiorum sp. nov. (type strain LMG 5906 T = API 184-2-84 T = CCUG 47000 T ), and several unnamed isolates. The DNA G+C content of members of the genus Kerstersia is between 61.5 and 62.9 mol%. On the basis of previously published DNA-DNA hybridization results and data from chemotaxonomic studies, it is proposed that Alcaligenes denitrificans Ruger and Tan 1983 be reclassified as Achromobacter denitrificans comb. nov.
Background
Schistosomiasis is the second-most widespread tropical parasitic disease after malaria. Various research strategies and treatment programs for achieving the objective of eradicating schistosomiasis within a decade have been recommended and supported by the World Health Organization. One of these approaches is based on the control of snail vectors in en-
demic areas. Previous field studies have shown that competitor or predator introduction can reduce snail numbers, but no systematic investigation has ever been conducted to identify snail microbial pathogens and evaluate their molluscicidal effects.
Methodology/Principal findings
In populations of Biomphalaria glabrata snails experiencing high mortalities, white nodules were visible on snail bodies. Infectious agents were isolated from such nodules. Only one type of bacteria, identified as a new species of Paenibacillus named Candidatus Paenibacillus glabratella, was found, and was shown to be closely related to P. alvei
through 16S and Rpob DNA analysis. Histopathological examination showed extensive bacterial infiltration leading to overall tissue disorganization. Exposure of healthy snails to Paenibacillus-infected snails caused massive mortality. Moreover, eggs laid by infected snails were also infected, decreasing hatching but without apparent effects on spawning. Embryonic lethality was correlated with the presence of pathogenic bacteria in eggs.
Conclusions/Significance
This is the first account of a novel Paenibacillus strain, Ca. Paenibacillus glabratella, as a snail microbial pathogen. Since this strain affects both adult and embryonic stages and causes significant mortality, it may hold promise as a biocontrol agent to limit schistosomiasis transmission in the field.
In the period from June to October 2008 we collected 500 slugs from the genus Arion in the area of Ljubljana and Prekmurje (Slovenia). By means of dissection we determined the presence of parasitic nematodes in slug cadavers. Identification of the nematodes was made by a molecular technique (PCR). In these slugs we did not find the parasitic nematode Phasmarhabditis hermaphrodita, however the presence of Alloionema appendiculatum in larger quantities was confirmed. The most infected was a Spanish slug, Arion
lusitanicus. In Petri dishes younger slugs showed a satisfactory mortality rate already on the fourth day after the
application of the nematode suspension. Unfortunately, we can not confirm with certainly that the nematode A.
appendiculatum undergoes a complete life cycle in A. lusitanicus, which is otherwise typical for Phasmarhabditis hermaphrodita.
We report the isolation of a bacterium from Galleria mellonella larva and its identification using genome sequencing and phylogenomic analysis. This bacterium was named Alcaligenes faecalis strain MOR02. Microscopic analyses revealed that the bacteria are located in the esophagus and intestine of the nematodes Steinernema feltiae, S. carpocapsae, and H. bacteriophora. Using G. mellonella larvae as a model, when the larvae were injected with 24,000 CFU in their hemocoel, more than 96% mortality was achieved after 24 h. Additionally, toxicity assays determined that 1 μg of supernatant extract from A. faecalis MOR02 killed more than 70% G. mellonella larvae 96 h after injection. A correlation of experimental data with sequence genome analyses was also performed. We discovered genes that encode proteins and enzymes that are related to pathogenicity, toxicity, and host/environment interactions that may be responsible for the observed phenotypic characteristics. Our data demonstrates that the bacteria are able to use different strategies to colonize nematodes and kill insects to their own benefit. However, there remains an extensive group of unidentified microorganisms that could be participating in the infection process. Additionally, a nematode-bacterium association could be established probably as a strategy of dispersion and colonization.
Under laboratory conditions, eight native bacterial isolates of Bacillus thuringiensis, were tested for their molluscicidal activity against the two land snails, Monacha cantiana (Montagu) and Eobania vermiculata (Muller) at a constant concentration (7x10 6 cfu/ ml) using three methods of application. Results revealed that all tested isolates applied via food or soil had no adverse effect on the tested land snails and resulted no mortality in M. cantiana and E. vermiculata as well. However, results clearly indicate the molluscicidal properties of the tested isolates and identify Bt-k64 ,Bt-B33 as well as Bt-W123 as potentially effective against M. cantiana and E. vermiculata when bacterial isolates were introduced to snails via food and soil simultaneously (concomitant treatment). For eight bacterial isolates, the percentage mortality ranged from 33% to 86.6% for the land snail, M. cantiana , while in the land snail, E. vermiculata the percentage mortality ranged from 6.6% to 53%. after four weeks of treatment B. thuringiensis designated as Bt-K64 was the most effective one against the two land snails, M. cantiana and E. vermiculata with percentage mortality 86.6% and 53.0% , respectively whereas, B. thurinigensis designated as Bt-B123 was the least toxic isolate with mortality 33% and 6.6% respectively. M. cantiana was more sensitive to the tested isolates than Eobania vermiculatum. Moreover, the molluscicidal activity of the three isolates of the bacterium, B. thurinigensis designated as Bt-W123 , Bt-B33 and Bt-K64 at three concentrations (1x10 6 , 1x10 7 and 1x10 8 cfu/ ml) against M. cantina under laboratory condition at 15ºC±3 and 60 ±5% RH. was undertaken. Isolate Bt –B33 and isolate Bt –W123 showed higher toxicity to the target snail than isolate Bt – K64 with mortality increasing with an increase in the concentrations of isolates as well as the period of exposure. At the highest concentration (1x10 8
This book documents and illustrates the major developments in the use of nematodes for biological control of insects and slugs. It has seven parts covering the morphology and taxonomy of all nematode groups used as biological control agents; entomopathogenic nematodes; entomophilic nematodes; slug-parasitic nematodes; predatory nematodes; fungal-feeding nematodes; and critical issues and research needs for future expansion of nematode use in biological control. Nematode biology, commercial production, formulation and quality control, application technology, strategy and safety are discussed. Separate chapters are devoted to the application of nematodes in different cropping systems and the efficacy of nematodes against specific pests such as plant parasitic nematodes and fungal pathogens.
In New Zealand, the European invasive slug Deroceras reticulatum is a pest in home gardens, horticulture, pastoral and arable agriculture. At present there are no biological control options available in New Zealand, unlike Europe, where the nematode parasite Phasmarhabditis hermaphrodita is sold in several countries under the trade name Nemaslug®. This paper describes the finding of a nematode parasitising D. reticulatum in New Zealand that was identified by morphological and molecular (18S rDNA sequencing) methods as P. hermaphrodita. The nematode was reared on slug cadavers and shown to infect and kill field-collected D. reticulatum. In mesocosm experiments with clover, the New Zealand P. hermaphrodita applied at the recommended commercial rate substantially increased plant density even under high slug pressure. © 2012 New Zealand Plant Protection Society (Inc.).
A nouvel Alicaligenes faecalis antibacterial-producing strain isolated from a Moroccan tannery waste
During an investigation on stool of Atherurus macrourus in Amol zoo, 16 parasites (14 females and
2 males) were collected. After fixing by Formalin, specimens cleared by lactophenol.
Their morphological andmorphometric characteristics were discussed elaborately.
Two new species of Steinernema (Rhabditida), Steinernema loci sp. n. and S. thanhi sp. n., were isolated from beach soil in Thanhhoa and Hatinh provinces in the northern part of Vietnam. A combination of morphological, morphometric and rDNA-RFLP features indicated the distinctness of both species from other Steinernema spp. Diagnostic characters of Steinernema loci sp. n. include: total body length (896-1072 μm), distance from anterior end to excretory pore (EP = 71-86 μm), tail length (66-83 μm), lateral field in mid-body with eight ridges (nine distinct lines) and rounded, broad and smooth anterior end of the third stage infective juvenile; first generation males were characterised by body width, spicule length and the ratio spicule length : anal body width (SW). Diagnostic characters of Steinernema thanhi sp. n. third stage infective juveniles include: total body length (720-960 μm), EP (68-84 μm), tail length (52-72 μm), lateral field in mid-body with eight ridges (nine distinct lines) and rounded, broad and smooth anterior end. Spicule length, ratio SW and arrangement of genital papillae characterised the first generation males.
The rhabditid nematode, Phasmarhabditis hermaphrodita is a lethal parasite of certain terrestrial gastropods and has been shown as a biocontrol agent under laboratory and field conditions. In Egypt, P. hermaphrodita was isolated for the first time from different species of terrestrial gastropods found associated with various crops at Aga and Mansoura districts of Dakhalia Governorate during the year 2000/2001. Females and dauer larvae (IJs) were described and illustrated based on the light microscope. Males are not found as this species seemed to be protandrous. PCR analysis confirmed nematode identification. The Egyptian isolate of P. hermaphrodita was found to be shorter and lower in width than the British isolate. V%, a, b and c parameters showed detectable variations between two isolates with values of 54%, 17.7, 4.28, 13.7 in the Egyptian isolate, and 51%, 19.5, 7.2 and 15.8 for the British isolate, respectively.
This 19-chapter book discusses the biology (including reproduction, life history, feeding preferences and sexual behaviour) of molluscs as pests of horticultural, field and fodder crops, and outlines the development of appropriate mechanisms for the control of these pests (mainly biological, cultural and chemical). Two chapters review progress towards the development of chemical control strategies, one addressing the toxicology of chemicals, the other the deployment of molluscicides in baits. These chapters also highlight the statistical and biological procedures for screening and evaluating molluscicides which are not a component of the standard procedure of mollusc control. A series of chapters focus on specific crop situations, providing a synopsis of the current pest status of gastropod species or species groups.
In the present investigation, emphasis was placed on the efficacy of lipopolysaccharide (LPS) extracted from the plant beneficial rhizobacterium Alcaligenes faecalis MSS8 for the induction of disease resistance by enhancing immune response in plants against phytopathogen Fusarium oxysporum f. sp. lycopersici (FOL). Characterization and proficiency of the rhizobacterial LPS for the induced defense response in plant against FOL was revealed by the gel activity staining of Peroxidase (PO) and Polyphenol oxidase (PPO) isoforms as compared to the untreated and only FOL treated plants. It has been observed that after 48 h of FOL challenge inoculation, in the LPS pretreated plant there was significant induction of PO and PPO isoforms. Furthermore, RT-PCR analysis for the study of relative gene expression of pathogenesis related (PR) proteins confirms the result of gel activity staining. It has been observed that expression level of LeChi9, LeGluB, LePR1a was significantly upregulated in LPS treated plants 24 h after FOL inoculation and maximum level was observed at 48 h as compared to the control and FOL treated plants. This clearly indicates the role of LPS extracted from A. faecalis as a potential biocontrol agent against fusarium wilt.
Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (> 32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Allionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras sp. compared to the resistant Arion sp. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes.
Terrestrial molluscs (Mollusca: Gastropoda) are important economic pests worldwide, causing extensive damage to a variety of crop types, and posing a health risk to both humans and wildlife. In South Africa, the climate is favourable for invasive European molluscs, especially in the Western Cape province, where there are mild, damp winters. One crop that is particularly targeted by the pests concerned is canola (Brassica napus), which is a winter arable crop that is commercially produced for its use in cooking, food processing, fertilisers, fuels, pet food, plastics, and animal feed. Molluscs on canola in the Western Cape province are currently controlled using chemical molluscicide pellets. These chemicals have the potential to adversely affect the environment and non-target organisms. The use of mollusc-parasitic nematodes is a possible environmentally-friendly alternative.
Current knowledge indicates that there are eight nematode families that associate with molluscs, including Agfidae, Alaninematidae, Alloionematidae, Angiostomatidae, Cosmocercidae, Diplogastridae, Mermithidae, and Rhabditidae. To date, Phasmarhabditis hermaphrodita is the only nematode that has been developed as a biological molluscicide. The nematode, which was commercially released in 1994 by MicroBio Ltd, Littlehampton, UK (formally Becker Underwood, now BASF) under the trade name Nemaslug®, is now sold in fifteen different European countries. Due to current legislation, Nemaslug® cannot be sold or used in South Africa. A survey was therefore conducted in the Western Cape province of South Africa to locate a local nematode isolate capable of causing mortality in invasive mollusc pests.
A total of 1944 slugs were collected from 12 different study sites. On the identification of slugs, they were dissected alive, and examined for internal nematodes. Nematodes were identified using morphological and molecular techniques (18S rRNA). Seven of the 12 sites had nematodes present, with 8% of the slugs being found to be infected with nematodes. Six
V
nematode species were identified, including Angiostoma margaretae, Angiostoma sp., Caenorhabditis elegans, a mermitid sp., and Phasmarhabditis spp. (SA3 and SA4). Of the six species mentioned, four were previously undescribed. The isolation of new Phasmarhabditis spp. indicates the importance of conducting further surveys of mollusc-parasitic nematodes in South Africa.
Nematodes isolated in the survey were tested for their ability to reproduce on decaying organic matter (consisting of dead frozen slugs), with results demonstrating that one of the nematodes, Phasmarhabditis sp. SA4, could complete its life cycle under such conditions. In addition, pathogenicity tests illustrated that Phasmarhabditis sp. SA4 caused significant mortality of the slug D. panormitanum.
Phasmarhabditis sp. SA4 was then fully described and characterised by the shape and length of the female tail, and by the presence of males. Phylogenetic analysis demonstrated that Phasmarhabditis sp. SA4 was placed in a monophyletic clade along with Phasmarhabditis sp. SA2, Phasmarhabditis papillosa, and the mollusc-parasitic nematode, Angiostoma dentiferum. The new species brings the total complement of the genus to seven species.
Phasmarhabditis sp. SA4 was then established in monoxenic cultures. Five bacterial isolates were isolated from the intestine of slug hosts, identified using 16S rRNA gene sequences, and their pathogenicity tested by means of injecting directly into the haemocoel of D. reticulatum, and monitoring the mortality over time. Kluyvera sp., which was found to cause the highest mortality rate among the slugs concerned, was chosen for monoxenic culturing. Cultures containing Phasmarhabditis sp. SA4 and Kluyvera sp. were optimised using temperatures ranging from 15°C to 25°C, with results showing that 15°C was the optimum growth temperature.
The impact of selected entomopathogenic nematodes and Phasmarhabditis hermaphrodita on the EuropeanUnion-protected slug Geomalacus maculosus and the sympatric Lehmannia marginata was investigated. There was no significant difference in mortality between slugs treated with nematodes and their controls. The presence of P. hermaphrodita in two G. maculosus cadavers may be the result of necromenic behaviour. This study constitutes the first record of P. californica in Europe.
Toxicity of several Bacillus thuringiensis preparations had been shown against the grey field slug Deroceras reticulatum (Muller) and the aquatic snail Biomphalaria alexandrina (Ehrenberg) by previous authors, letting view a method of microbiological control of gastropods. Therefore, the toxicities of some of these products and of several unformulated strains, having different insecticide activities, were studied on three species of slugs: D reticulatum, Arion distinctus Mabille and Limax valentianus Ferussac, and compared to molluscicide products of reference. Amongst the products tested, two contained the β-exotoxin (Bitoxibacillin 5 and the surnatant H1). However, no strain of Bacillus showed to be toxic for the three slug species. Only the molluscicide products confirmed their efficiency against the slugs.
African giant snails (Archachatina marginata ovum Pfeffer, 1858; A. marginata saturalis Philippi, 1849 and Achatina achatina Linne, 1758) were examined for the occurrence of Rhabditis axei Cobbold, 1884. Differences in parasite intensity between size groups of snails were highly significant (P < 0.05) for the 3 species compared. The mean egg and larval output per gram of faeces was higher in larger snails. The mean intensity of the nematode eggs excreted was higher (P < 0.05) than the mean of larval output for the 3 species of snails. The distribution of R. axei within the snail hosts revealed site preferences. They are mostly (88 %) located in the rectum of the snails.
The efficiency of six bacterial strains to kill cotton leaf worms (Spodoptera littoralis), was investigated. These bacterial strains were originally isolated from dead spiny cotton bollworms (Erias insulana). Leaf cotton worms were fed for four days on castor bean (Ricinus communis) leaves (an alternative to cotton leaves), which were soaked for 30 minutes in suspensions of four-days old bacterial cultures. The bacterial species were very offensive and its mortality was relatively high, (about 20-60 % of leaf cotton worms were killed). Preliminary characterization of the bacterial strains revealed that the six strains were gram positive, rod-shaped, and sporeforming with variable morphology. Based on cultural characteristics and phenotypic tests (e.g., salt tolerance, heat tolerance, and some enzymatic activities), the spore-forming rods were tentatively related to genus Bacillus. According to 16SrRNA genes analysis, the gram positive bacteria were classified under genus Bacillus with gene sequence similarity up to 96-100 %. The bacterial species are distributed in six bacillus species, namely: Bacillus anthracis, Bacillus cereus, B. fusiformis, B. pseudomycoides, B. subtilis, and B. thuringiensis. In conclusion, the bacterial species identified in this study are good candidates for biological control of the destructive cotton pests (leaf worms and spiny or pink bollworms) in Egypt.
The decreasing productivity of silkworm cocoon is caused by attack of bacteria, fungi, protozoa and virus. The research was aimed to isolate of suspected pathogenic and biological control bacteria from damage and healthy pupae of golden silkworm (Cricula trifenestrata Helfer) and to do in vivo test of the bacteria toward 4th instar larvae of C. trifenestrata and mulberry silkworms, Bombyx mori Linn. The isolate UPC 60 was suspected as pathogenic bacteria due to cause the mortality of silkworms larvae. The bacteria UPC 40 and UPC 71 were selected as biological control bacteria which had capability to reduce the mortality of silkworms about 56.67 and 43.33%, respectively. Based on 16s RNA gene the bacteria UPC 40 had 97% identity with Alcaligenes faecalis, bacteria UPC 60 had 97% identity with Aeromonas dhakensis and bacteria UPC 71 had 98% identity with Pseudomonas stutzuri. The biological control bacteria showed antagonistic activity toward the pathogenic bacteria. The outcomes of the research that the biological control bacteria expected to be used to reduce pathogenic bacteria in the maintenance of silkworm so that the production and quality of silk yarn can be improved.
Alloionema appendiculatum is a common juvenile parasite of many terrestrial molluscs. Its 3rd stage juveniles (dauers) invade the foot muscle of snails and slugs. Dauer juveniles develop into 4th stage juvenile, which then leave the host. Later they mature and reproduce in the soil. A population of A. appendiculatum was isolated from infected individuals of the invasive slug Arion vulgaris (= A. lusitanicus) collected in the city of České Budějovice, Czech Republic, and was designated as the AL strain. This nematode is also able to reproduce on pig kidney in laboratory culture. Our isolate was compared primarily with the description published by Mengert (1953) and in some aspects with the original description published by Schneider (1859). Insufficient morphology, morphometrics, phylogeny and an absence of pictorial material encouraged us to re-describe this frequent slug parasite and to add some new information on its life-cycle. The species is characterised by the absence, in adults, of ridges in the lateral fields. At this life stage the stoma is short, narrow and approximately twice as long as it is broad. Fourth stage female juveniles produce a mucus-like substance from the phasmids. Males possess six pairs of papillae and a single inconspicuous papilla. The nematode has both parasitic and saprophytic lifecycles. Parasitic adults are bigger than the saprophytes and have a thick, digitate tail, whereas the smaller saprophytic generation has a filiform tail. The species is amphimictic, displaying a higher proportion of females, but males are frequent.
The Giant African snail (Achatina fulica) is a major pest in tropical countries. Current control methods involve the use of slug pellets (metaldehyde) but they are ineffective, therefore new methods of control are needed. We investigated whether A. fulica is susceptible to the gastropod parasitic nematode Phasmarhabditis hermaphrodita, which has been developed as a biological control agent for slugs and snails in northern Europe. We exposed A. fulica to P. hermaphrodita applied at 30 and 150 nematodes per cm(2) for 70 days and also assessed feeding inhibition and changes in snail weight. We show that unlike the susceptible slug species Deroceras panormitanum, which is killed less than 30 days of exposure to P. hermaphrodita, A. fulica is remarkably resistant to the nematode at both doses. Also P. hermaphrodita does not reduce feeding in A. fulica nor did it have any effect on weight gain over 70 days. Upon dissection of infected A. fulica we found that hundreds of P. hermaphrodita had been encapsulated, trapped and killed in the snail's shell. We found that A. fulica is able to begin encapsulating P. hermaphrodita after just 3 days of exposure and the numbers of nematodes encapsulated increased over time. Taken together, we have shown that A. fulica is highly resistant to P. hermaphrodita, which could be due to an immune response dependent on the snail shell to encapsulate and kill invading parasitic nematodes.
Copyright © 2015. Published by Elsevier Inc.
Sexual outcrossing is costly relative to selfing and asexuality, yet it is ubiquitous in nature, a paradox that has long puzzled evolutionary biologists. The Red Queen Hypothesis argues that outcrossing is maintained by antagonistic interactions between host and parasites. Most tests of this hypothesis focus on the maintenance of outcrossing in hosts. The Red Queen makes an additional prediction that parasitic taxa are more likely to be outcrossing than their free-living relatives. We test this prediction in the diverse Nematode phylum using phylogenetic comparative methods to evaluate trait correlations. In support of the Red Queen, we demonstrate a significant correlation between parasitism and outcrossing in this clade. We find that this correlation is driven by animal parasites, for which outcrossing is significantly enriched relative to both free-living and plant parasitic taxa. Finally, we test hypotheses for the evolutionary history underlying the correlation of outcrossing and animal parasitism. Our results demonstrate that selfing and asexuality are significantly less likely to arise on parasitic lineages than on free-living ones. The findings of this study are consistent with the Red Queen Hypothesis. Moreover, they suggest that the maintenance of genetic variation is an important factor in the persistence of parasitic lineages.This article is protected by copyright. All rights reserved.
The level of cellulolytic activity in different areas of the gut of the terrestrial slug Arion ater was assayed at different temperatures and pH values. To do this, crude gut proteins were isolated and assayed using modified dinitrosalicylic acid reducing sugar assay. Crude proteins sample were also separated and cellulolytic activity identified using in gel CMC zymography and esculin hydrate activity gel assays. pH and temperature profiling revealed optimum cellulolytic activity between pH5.0 and 6.0 for different gut regions and retention of up to 90% of activity at temperatures up to 50°C. Zymograms and activity gels revealed multiple endoglucanase and β-glucosidase enzymes. To further investigate the source of this cellulolytic activity bacterial isolates from the gut were tested for endoglucanase and β-glucosidase activity using growth plate assays. 12 cellulolytic microbes were identified using 16S rDNA gene sequencing. These include members of the genera Buttiauxella, Enterobacter, Citrobacter, Serratia, Klebsiella. Gut metagenomic DNA was then subjected to PCR, targeting a 400bp region of the 16SrDNA gene which was subsequently separated and individuals identified using DGGE. This identified members of the genera Citrobacter, Serratia, Pectobacterium, Acinetobacter, Mycoplasma, Pantoea and Erwinia. In summary, multiple glycoside hydrolase enzymes active over a broad range of temperature and pH values in a relatively under studied organism were detected, indicating that the gut of A. ater is a viable target for intensive study to identify novel carbohydrate active enzymes that may be used in the biofuel industry.
Bacteria were isolated from the crop, digestive glands and salivary glands of the field slug Deroceras reticulatum (Muller). The digestive gland was the most densely populated organ with about x3 and x400 more bacterial colony forming units (CFU) mg-1 of tissue than in the crop and salivary glands, respectively. Growth on plate-count agar incorporating gelatin showed that 34, 16 and 3% of the total numbers of CFU present in the salivary gland, digestive gland and crop, respectively, secreted extracellular proteinases that degraded gelatin. Administration of antibiotics (chloramphenicol or tetracycline plus chlortetracycline) to slugs in an artificial diet reduced the numbers of total and proteolytic CFU present by 98-99%. Although proteinase-secreting bacteria were present in all organs studied, only the crop possessed significantly greater proteolytic activity (x3 at pH 8.0) when slugs were not fed on diet containing antibiotics.
In a soil bioassay, adult Deroceras reticulatum (Stylommatophora: Limacidae) and three different weight-classes of young Arion lusitanicus (Stylommatophora: Arionidae) were exposed to a single dosage (170 dauer larvae per g of soil) of the nematode Phasmarhabditis hermaphrodita monoxenically associated with the bacterium Moraxella osloensis. Groups of 10 slugs were continuously exposed to nematodes for 4 days, and then transferred individually to Petri-dishes containing a disc of Chinese cabbage as food. Food consumption - measured by image analysis - and slug mortality were recorded daily for 10 days. Food consumption was inhibited in both slug species tested. D. reticulatum stopped feeding 6 days after the start of nematode treatment, while all A. lusitanicus continued to feed. However, in the three weight-classes of A. lusitanicus (0.15 g, 0.24 g, 0.45 g), food consumption was reduced by at least 50 %. The greatest reduction in feeding, nearly 90 %, was noted in the smallest A. lusitanicus. The nematodes successfully killed D. reticulatum but were less efficient at killing young A. lusitanicus. At the end of the experiment, mortality was highest in D. reticultatum (98 %) and the smallest weight-class of A. lusitanicus (47 %). There was almost no mortality in the largest weight-class of A. lusitanicus treated with nematodes. P. hermaphrodita associated with M. osloensis can thus be considered as a biological control agent for young stages of A. lusitanicus for its effect as a feeding inhibitor, rather than for its ability to kill the slugs.
A nematode, Phasmarhabditis hermaphrodita, known to be associated with slugs but not previously thought to be parasitic, was shown to be a parasite capable of killing the pest slug Deroceras reticulatum. The parasite infects slugs in the area beneath the mantle surrounding the shell, causing a disease with characteristic symptoms, particularly swelling of the mantle. Infection leads to death of the slug, usually between seven and 21 days afterwards. The nematode then spreads and multiplies in the cadaver. In an experiment where individual D. reticulatum were exposed to different numbers of P. hermaphrodita, a significant positive relationship was found between nematode dose and slug mortality. In two experiments on host range, the nematode was found to infect and kill all pest slug species tested: Deroceras caruanae, Arion distinctus, Arion silvaticus, Arion intermedius, Arion ater, Tandonia sowerbyi and T. budapestensis, in addition to D. reticulatum.
Pairs and single adults of the terrestrial snail Cernuella virgata were exposed to various densities of the rhabditid isolate R954 during breeding experiments in the laboratory. Fecundity and viability of C. virgata breeding in nematode-free and in nematode-infested soil was compared. Exposure to R954 significantly reduced the number of eggs produced by C. virgata and the survival of adult snails. Differences in nematode densities (50, 100 and 150 nematodes cm -2 ) had no effect on egg production or snail survival. Snail eggs laid during the breeding experiments were fertile and young hatched after 2-3 weeks. R954 was not able to penetrate eggs of C. virgata but nematodes were observed inside the hatchlings and caused high mortality.
The slug, Deroceras reticulatum (Stylommatophora: Limacidae), was exposed to different concentrations of infective dauer juveniles of the rhabditid nematode Phasmarhabditis hermaphrodita, in a two-stage bioassay, at 10°C. Slugs were exposed in groups of 10 or 12 to nematodes in plastic boxes filled with soil aggregates for 3 or 5 days and then transferred individually to petri dishes each containing a disk of Chinese cabbage leaf as food. Subsequently, slug food consumption and survival were measured for 10 to 13 days. Models were developed to describe the way that exposure to the nematode caused inhibition of slug feeding followed by death. Both effects were related to nematode concentrations and time after exposure to the nematode. Following exposure to high concentrations (300,000 dauer juveniles per box), slugs were killed rapidly, within a few days after the end of the exposure period. Following exposure to low concentrations of nematodes (7000 or 15,000 per box), substantial numbers of slugs survived until the end of the bioassay, but feeding activity by these slugs was strongly inhibited. It is suggested that inhibition of slug feeding is important for the success of this nematode as a biocontrol agent.
Nutrient agar, albumin agar and azocoll agar media were used to screen soil proteolytic bacteria from paddy fields. Proteolytic activity was compared by evaluating extracellular hydrolytic activities toward benzyloxycarbonyl-l-phenylalanyl-l-leucine (z-FLase), benzyloxycarbonyl-l-phenylalanyl-l-tyrosyl-l-leucine (z-FTLase) and casein (caseinase).The average z-FLase activity (10.6 nKat g−1 protein) of bacteria capable of gelatin liquefaction isolated from azocoll nutrient agar (ANB/GL, 37 isolates) was 33.7 times that of bacteria capable of gelatin liquefaction isolated from nutrient agar (NB/GL, 24 isolates) and 6.7 times that of actinomycetes capable of gelatin liquefaction isolated from albumin agar (AA/GL, 33 isolates), the z-FTLase activity (24.5 nKat g−1 protein) was 41.3 times that of NB/GL and 6.2 times that of AA/GL, and caseinase activity (34.0 nKat g−1 protein) was 27.8 times that of NB/GL and 14.0 times that of AA/GL.Therefore, azocoll nutrient agar was an effective isolation medium for screening soil bacteria with extremely high extracellular protease activities.
Bacterial strain possessing both bacteriostatic and fungistatic activity (biocontrol activity) against pathogens of cyclamen (Cyclamen sp.) was isolated from the soil in Gifu Prefecture, Japan, and characterized with respect to its taxonomic and biocontrol properties. The sequence of its 16S rRNA gene, morphology, biochemistry, and fatty acid composition demonstrated that it is a strain most closely related to Alcaligenes faecalis subsp. faecalis LMG 1229(T). The isolate was named A. faecalis strain AD15. A. faecalis AD15 produced hydroxylamine at maximum yields of 33.3±1.7 mg/L after 16 h cultivation in LB medium and 19.0±0.44 mg/L after 19 h cultivation in synthetic medium. Moreover, minimum inhibitory concentrations of hydroxylamine against the cyclamen pathogens Pantoea agglomerans and Colletotrichum gloeosporioides were 4.20±0.98 and 16.5±0.67 mg/L. These results indicated that the biocontrol activity of strain AD15 might be attributed to hydroxylamine, a metabolite in the culture medium, and it had the potential for biopesticide application.
Robbie Rae, Jamie Robertson and Mike Wilson, outline the problems that slugs cause in many temperate crops and introduce new treatment strategies for their control.
The probit method, i.e., the reduction of a sigmoid response curve to a straight line by means of a transformation of the responses based on a normal integral, was invented by Fechner. It has been used in the method of constant stimuli of psychophysics. This volume gives a systematic account of the theory and practice of the method. The first 10 chapters offer a development of the practical and computational aspects. Appendix I gives a detailed description of a systematic arrangement of the computations for the simplest types of analysis. The theory of the probit method is derived from principles as outlined in Appendix II. 22-item bibliography. (PsycINFO Database Record (c) 2012 APA, all rights reserved)