Authors: John Hart and Keith Davies

Abstract

In this study using Caenorhabditis elegans, we have been able to suppress (>60%) and enhance (>40%) fecundity (number of offspring) while extending lifespan by a fifth, by administering synthetic peptides to the aqueous medium in which the nematodes were maintained. Untreated control adults fed live bacteria had significantly more offspring (17 vs 10 larvae each) than those fed dead bacteria. Average lifespan and time for 50% of the worms to die were the same at approximately 10 days, but there was a significant difference in terms of 100% mortality (28 vs 19 days). A reduction in fecundity of 30-40% occurred when a 14-mer peptide, EPL030, was administered to the worms' aqueous medium. The effect was dose-dependent across the range 0.1-10 μM day–1 of medium, but since the worms were fed live bacteria interpretation was problematic: was the effect direct or indirect? However, the anti-fecundity effect was reproduced in worms fed dead bacteria, when the test compound was administered at 1 μM day–1 of aqueous medium. The mean number of larvae produced in three groups: untreated controls, EPL030 and EPL001 (an anagrammatical version of EPL030 used as a comparator), were, respectively, 17, 6 (−64%) and 24 (+43%). Average lifespans were 8.7, 10.7 (+23%) and 10.3 days (+18%). Fluorescence localisation studies using a close analogue of the fecundity-suppressing EPL030 revealed a distribution that was generalised and uninformative. The fecundity-enhancing EPL001 concentrated in the genital tract. Caenorhabditis elegans is a potentially useful testbed for fecundity and lifespan studies using exogenous agents. The use of an aqueous medium and dead bacteria as food simplifies both the protocol and interpretation of results.

In: Nematology

Abstract

Pasteuria penetrans isolates sampled from different geographical areas were characterised both for the heterogeneity of the endospore surface using monoclonal antibodies and for the ability of spores to attach to different isolates of Meloidogyne spp. The efficacy of these different Pasteuria isolates as biological control agents was tested in a glasshouse experiment with M. incognita from Senegal on Acacia holosericea. The immunoprofiles divided the P. penetrans isolates broadly differently from the attachment tests. Isolate PP16 from Senegal was associated with better seedling development of M. incognita-inoculated A. holoceria than were other isolates. Substantial variation in root and shoot biomass was not related to the observed variation in spore attachment tests. The difficulties involved in obtaining consistent biological control with Pasteuria are discussed in relation to the high degree of variability of this bacterium. Diversité chez l'hyperparasite bactérien Pasteuria penetrans en relation avec le contrôle du nématode Meloidogyne spp. sur Acacia holosericea - Des isolats de Pasteuria penetrans provenant de différentes régions géographiques ont été caractérisés en ce qui concerne, d'une part l'hétérogénéité de la surface des endospores - par utilisation d'anticorps monoclonaux -, d'autre part la capacité des spores à s'attacher à différentes souches de Meloidogyne spp. L'efficacité de ces différents isolats de P.penetrans en tant qu'agent de contrôle a été testée lors d'une expérience en serre utilisant un M. incognita d'origine sénégalaise et Acacia holosericea. Les isolats de P.penetrans ont montré des différences dans les profils immunologiques, différences non corrélées avec celles observées lors des tests d'attachement. En comparaison avec d'autres isolats, l'isolat PP16 provenant du Sénégal était associé à une meilleure croissance des plants de A. holosericea infestés par M. incognita. La variabilité importante touchant les biomasses aérienne et souterraine ne montrait aucune relation avec les différences observées lors des tests d'attachement. Les difficultés rencontrées pour obtenir un contrôle valable à l'aide de P.penetrans sont discutées en tenant compte du degré important de variabilité chez cette bactérie.

In: Nematology

The molecular interactions between hosts and parasites is an active area of research, and the parasitism of root-knot nematodes, obligate parasites of plants, by the hyper-parasitic bacterium Pasteuria penetrans offers a model by which to investigate aspects of innate immunity. Using a pouch system we were able to demonstrate by PCR, infection of Meloidogyne incognita 4 days prior to any microscopic observations of parasitism. The pouch system, although not strictly axenic, offered a relatively clean, flexible approach with a greatly reduced number of contaminating microbial species than in any soil-based system, whereby the early stages of nematode infection could be manipulated and controlled.

In: Nematology

Abstract

This is the first report of endospores of Pasteuria penetrans and P. nishizawae isolates binding to juveniles of Meloidogyne chitwoodi and M. fallax. The patterns of endospore attachment to M. chitwoodi and M. fallax compared with the related M. hapla suggest that there are differences amongst these three temperate root-knot nematode species. Intraspecific variability in attachment of P. penetrans to juveniles of M. chitwoodi was demonstrated.

In: Nematology

Three experiments were conducted to compare the attraction and repulsion of second-stage juveniles (J2) of the root-knot nematode, Meloidogyne graminicola, to rice plants supplied with different forms of nitrogen. The rice plants were hydroponically grown in a full nutrient solution containing different concentrations (0.1 or 10.0 mM) of nitrate (NO3 ) or forms of nitrogen supply (2.85 mM calcium nitrate (Ca(NO3)2), ammonium nitrate (NH4NO3) or ammonium chloride (NH4Cl)) for 2 weeks. Five rice plants were placed with their roots in one corner of a Perspex X or Y-chamber partly filled with agar and J2 were inoculated onto the chambers. The data show that J2 of M. graminicola were significantly attracted towards the roots of rice plants grown in hydroponics containing 0.1 mM NO3 and 2.85 mM Ca(NO3)2, but repelled by 10.0 mM NO3 , 2.85 mM NH4NO3 and NH4Cl. The results suggest that the application of ammonia-based nitrogen fertiliser to the rice nursery bed may interfere with nematode attraction and thus reduce invasion, and the application of chemical nitrification inhibitors to rice nursery beds may decrease nematode invasion.

In: Nematology

Abstract

Pseudomonas oryzihabitans, a bacterium associated with the entomopathogenic nematode Steinernema abbasi, was evaluated for its potential to colonise roots and thereby control a field population of root-knot nematodes. Immunological techniques were developed to detect root colonisation of P. oryzihabitans on tomato roots using a specific polyclonal antibody raised against vegetative bacterial cells. In vitro, bacterial cell filtrates were also shown significantly to inhibit juveniles hatching. In a glasshouse pot experiment, there were 22 and 82% fewer females in roots of plants treated with suspensions containing 103 and 106 cells ml−1 of P. oryzihabitans, respectively. In addition, there were significantly fewer egg masses produced; however, the numbers of eggs per egg mass did not differ significantly. The relationship between root colonisation and nematode control is discussed.

In: Nematology

Abstract

Comparisons of the growth of Pasteuria penetrans in adult root-knot nematode females infected with P. penetrans dissected from the roots of tomato plants were undertaken using bright-field and scanning electron microscopy. Samples of infected females were nutritionally compromised by maintaining them in sterile saline at 30°C for different periods of time following their removal from the root system. Observations of these females maintained in saline revealed a series of growth stages of Pasteuria hitherto not documented, consisting of rhizoids, rod-like bacilli and granular masses. A new life-cycle for Pasteuria is described consisting of three phases: Phase I: attachment and germination; Phase II: rhizoid production and exponential growth; and Phase III: sporogenesis. These newly observed stages of the life cycle show a high degree of similarity to the developmental stages seen in other Bacillus spp.

In: Nematology

Abstract

The conclusions of a collaborative study of the occurrence and importance of root-knot nematodes (RKN, Meloidogyne spp.) and of their control agent, Pasteuria penetrans, in parts of Europe, Africa, South America and the Caribbean are presented. Rootknot nematodes were estimated to reduce the yields of a wide range of horticultural crops by > 25% in Ecuador, Malawi and Tanzania, and by ca 10% in Trinidad and Tobago. The greatest proportion of infected crops were observed in Ecuador (205 of 207) and the least in Trinidad and Tobago (70 of 174). The mean gall index was greatest in Ecuador (5.5). Levels of galling were least in Senegal (1.6), even though 89% of crops were infested and virulent M. mayaguensis was widespread. In all countries, M. incognita and M. javanica were the most abundant species, but M. hispanica occurred widely in Burkina Faso, even in newly cultivated areas in the Sahile. Several new esterase phenotypes were detected, especially in Ecuador and Malawi. Juveniles (J2) collected from the soil during the surveys were examined for attached spores of P.penetrans. It was widespread (20 to 60% of RKN populations), except in Malawi and Tanzania (< 10% were infected), and was found for the first time in Crete (Greece). Generally, < 50% of the J2 carried spores. The occurrence of P.penetrans was sometimes correlated with soil type e.g., in Senegal it was least frequent in sandy soils. Laboratory assays of the binding of spores of isolates of P.penetrans to populations of RKN indicated large variations in specificity and substantial interactions; differences between populations within a species of RKN were sometimes almost as great as those between species. In microplot trials in which an "exotic" isolate of P.penetrans was introduced (ca 103 spores per g soil), its incidence was not increased by increasing the frequency or intensity of the growing of RKN-susceptible crops. However, in two such trials at sites in Tanzania and Ecuador naturally infected with P.penetrans, there were large increases in the proportions of spore-encumbered J2 (up to 100% encumbered) and in the yields of spores (up to 3.3 × 106 spores per mg dry root) in those plots amended with an "exotic" isolate. In these plots, numbers of J2 in the soil were decreased and damage by RKN was suppressed; gall indices were decreased (from > 8 to < 3) and yields were increased (by up to 30%). No such changes were observed in the unamended control plots. Increased suppression of RKN was also observed in a field trial, even in plots where RKN-susceptible and non-host crops were alternated. Increased suppression following amendment with the "exotic" isolate of P.penetrans was not observed at sites not previously infected with P.penetrans. Regression analysis of the results from the microplot and field trials indicated that tomato yields were decreased by > 5% for every increase of one in the gall index. Yields were increased by alternating tomato with leguminous crops in some trials, but not in others. It is proposed that, in natural infections, mutual selection produces a dynamic balance between the P.penetrans and the RKN whereby levels of infection are rarely suppressive. However, the introduction of an "exotic" isolate of P.penetrans, with a different attachment profile, can disturb this balance, resulting in a greatly increased proportion of infected J2 and females, increased yields of spores and more suppression of RKN populations. Importance des nématodes à galles tropicaux (Meloidogyne spp.) et facteurs affectant l'utilité de Pasteuria penetrans, agent de contrôle biologique - Ce travail présente les conclusions d'une étude, menée en collaboration par plusieurs équipes de recherche, sur la présence et l'importance des nématodes phytoparasites du genre Meloidogyne et de leur parasite bactérien, Pasteuria penetrans, dans certains pays d'Europe, d'Afrique, d'Amérique du Sud et des Caraïbes. Les réductions de rendement de cultures maraîchères très diverses dues à ces nématodes atteignent 25% en Equateur, Malawi et Tanzanie, et 10% à Trinidad et Tobago. La plus forte proportion de parcelles infestées a été rencontrée en Equateur (205 sur 207) et la plus faible à Trinidad et Tobago (70 sur 174). C'est en Equateur que l'indice de galles moyen (égal à 5,5) était le plus élevé. Même si cet indice était faible en moyenne au Sénégal (1,6), 89% des cultures étaient infestées dans ce pays, en grande partie par l'espèce virulente M. mayaguensis. M. incognita et M. javanica sont les plus répandues dans tous les pays prospectés. Cependant, M. hispanica est très répandu au Burkina Faso, même dans des zones récemment cultivées en maraîchage en région sahélienne. Plusieurs phénotypes estérasiques nouveaux ont été détectés, spécialement en Equateur et au Malawi. Les juvéniles de second stade (J2) extraits des échantillons de sol collectés lors des prospections ont été examinés pour détecter la présence de spores de P.penetrans sur leur cuticule. Trouvé pour la première fois en Crète (Grèce), P.penetrans est très répandu dans les autres pays prospectés, infestant 20 à 60% des populations de Meloidogyne spp., sauf au Malawi et en Tanzanie où moins de 10% des populations sont atteintes. Le plus souvent, moins de 50% de J2 portent des spores bactériennes. Le taux de parasitisme des J2 par P. penetrans est influencé par les types de sols, comme par exemple au Sénégal où il est très faible dans les sols sableux grossiers. Des expériences en laboratoire portant sur l'attachement de spores de divers isolats de P. penetrans à des populations de Meloidogyne spp. ont révélé une grande variation de la spécificité et des interactions; les différences observées pour diverses populations d'une même espèce de Meloidogyne sont parfois presque aussi marquées que celles observées entre espèces. L'incidence parasitaire d'un isolat "exotique" de P. penetrans introduit dans des microparcelles (ca. 103 sporespar g. de sol) n'a pas été accrue par l'augmentation de la fréquence ou de la densité de plantation des cultures sensibles à Meloidogyne spp. employées. Cependant, dans deux microparcelles natullement infestées en P.penetrans, situées en Equateur et en Tanzanie, l'introduction d'un isolat "exotique" s'est traduite par un accroissement de la proportion de J2 infestés (jusqu'à 100%) et de la production de spores ((jusqu'à 3, 3 × 106 spores par mg [poids sec] de racines), d'une diminution de la population de J2 dans le sol, et d'une disparition des dégâts racinaires; les indices de galles moyens ont diminué (de plus de 8 à moins de 3) et les rendements des cultures ont augmenté (jusqu'à 30%). De tels changements n'ont pas été observés dans des sols non amendés en isolats "exotiques" de P.penetrans. Un meilleur contrôle des populations de Meloidogyne spp. a également été observé au champ, même lorsque la séquence culturale alternait des cultures sensibles et des cultures non-hôtes du nématode. Dans des parcelles non infectées en P.penetrans, la baisse des populations de Meloidogyne spp. n'a pas été observée après introduction d'un isolat "exotique" de la bactérie. Des analyses de régression portant sur les données obtenues en microparcelles ou au champ montrent que les rendements en tomate diminuent de plus de 5% chaque fois que l'indice de galle augmente d'une unité. Ces rendements ont parfois été améliorés lorsque des cultures de légumineuses alternaient les cultures de tomate. Ainsi, sur la base des analyses nématologiques et agronomiques faites en fin de cycles culturaux, il est suggéré que, dans les cas d'infestations naturelles en P.penetrans, des sélections mutuelles entraîneraient un équilibre dynamique entre les populations de la bactérie et celles du nématode, représentatif d'une densité-dépendance retardée. En revanche, l'introduction inondative d'isolats "exotiques" de P.penetrans, aux propriétés parasitaires différentes de celles des populations natives, pourraient rompre temporairement cet équilibre en faveur d'un accroissement de la proportion de nématodes (J2 et femelles) infestés et de la production de spores bactériennes, ainsi qu'un meilleur contrôle des populations de Meloidogyne spp. La capacité des populations de P.penetrans à survivre dans les sols et à contrôler durablement les populations de Meloidogyne spp. dépendraient de la spécificité entre les organismes, des propriétés des sols et des systèmes de culture.

In: Nematology