Understanding the movement of root-knot nematodes encumbered with or without Pasteuria penetrans

Pasteuria penetrans is a naturally occurring bacterial parasite of plant parasitic nematodes showing satisfactory results in a biocontrol strategy of root-knot nematodes (Meloidogyne spp.). The endospores attach to the outside nematode body wall (cuticle) of the infective stage second-stage juveniles (J2) of Meloidogyne populations. Optimal attachment level should be around 5–10 endospores per juvenile, as enough endospores will initiate infection without reducing the ability of the nematode to invade roots. Greater than 15 endospores may disable the nematode in its movements, and invasion may not take place. In this research, evidence is provided that P. penetrans spores disturbed the nematode forward movement by disorganising the nematode's head turns. The results based on Markov chain and Cochran probability model show that even a low number of 5–8 spores of P. penetrans attached to the nematode cuticle have a significant impact on that movement, which plays a role in nematode locomotion.     

Fitting the negative binomial distribution to Pasteuria penetrans spore attachment on root-knot nematodes and predicting probability of spore attachments using a Markov chain model

Pasteuria penetrans controls root-knot nematodes (Meloidogyne spp.) either by preventing invasion or by causing female sterility. The greatest control of P. penetrans occurred when an appropriate quantity of P. penetrans spores attached to the cuticle of a juvenile nematode. The number of spores attaching to juveniles within a given time increased with increasing the time of exposure to spores. Based on this, numbers of encumbered nematodes were recorded 1, 3, 6 and 9 h after placing nematodes in standard P. penetrans spore suspensions. From the count data obtained, P. penetrans attachment was modelled using the Poisson and negative binomial distributions. Attachment count data were observed to be overdispersed with respect to high numbers of spores attaching on each J2 at 6 and 9 h after spore application. It was concluded that the negative binomial distribution was shown to be the most appropriate model to fit the observed data-sets considering that P. penetrans spores are clumped; this could be further refined with a Markov process.     

Observations on the pathogenicity of Pasteuria penetrans, a parasite of root-knot nematodes

A simple test was used to determine whether or not Pasteuria penetrans spores would attach to 17 species of nematodes. All susceptible individuals had spores attached to their cuticles after 24 h of gentle agitation in suspensions containing 10⁵spores/ml. Spores of P. penetrans from six populations of Meloidogyne only adhered to species of Meloidogyne and they adhered in greatest numbers to the species from which they had been originally isolated. Sonication of spores from infected females increased attachment but the effect was dependent on pH and whether the test was conducted in tap or distilled water. Invasion of tomato roots was reduced by up to 86% when, rather than using healthy juveniles, second-stage juveniles bearing 15 or more spores were added to soil at high densities (1000 or 3000/plant); at low densities (500/plant) invasion was not significantly affected. The rate of development of M. incognita juveniles infected with P. penetrans was slower than that of healthy juveniles. The numbers of second-generation of M. incognita were reduced by 82–93% when juveniles encumbered with 1–15 spores were added to soil instead of those bearing no spores. Pasteuria penetrans populations differed in their aggressiveness and when juveniles encumbered with the same number of spores from two populations were added to soil there were differences in the numbers of females that became infected. The implications of these results for the development of P. penetrans as a biological control agent are discussed.     

Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

Effects of Pseudomonas fluorescens strain UTPF5 on the mobility,mortality and hatching of root-knot nematode Meloidogyne javanica

The root-knot nematode Meloidogyne spp. includes important plant pathogens worldwide. This study has considered nematode Meloidogyne javanica second stage larvae activity in the extracts of Pseudomonas fluorescens strains UTPF5 and cytotoxic effect of the strain on the nematode. The movement of second stage larvae of nematodes in water agar medium at four concentrations of bacterial extracts and second stage larvae mortality rate of hatching nematode and bacterial strains in vitro were affected. Different concentrates of the strain UTPF5 effect nematode larvae movement and disposal of the same. Bacterial extraction kills almost 100% of the larvae hatching after 24?h and a complete ban on egg hatch of biocontrol nematodes and nematode indicated that root-knot nematode larvae movement on the right attract the bacteria P. fluorescens to extract in the first place.     

Host Range and Ecology of Isolates of Pasteuria spp. from the Southeastern United States

Isolates of Pasteuria penetrans were evaluated for ecological characteristics that are important in determining their potential as biological control agents. Isolate P-20 survived without loss of its ability to attach to its host nematode in dry, moist, and wet soil and in soil wetted and dried repeatedly for 6 weeks. Some spores moved 6.4 cm (the maximum distance tested) downward in soil within 3 days with percolating water. The isolates varied greatly in their attachment to different nematode species and genera. Of five isolates tested in spore-infested soil, three (P-104, P-122, B-3) attached to two or more nematode species, whereas B-8 attached only to Meloidogyne hapla and B-I did not attach to any of the nematodes tested. In water suspensions, spores of isolate P-20 attached readily to M. arenaria but only a few spores attached to other Meloidogyne spp. Isolate P-104 attached to all Meloidogyne spp. tested but not to Pratylenchus scribneri. Isolate B-4 attached to all species of Meloidogyne and Pratylenchus tested, but the rate of attachment was relatively low. Isolate P-Z00 attached in high numbers to M. arenaria when spores were extracted from females of this nematode; when extracted from M. javanica females, fewer spores attached to M. arenaria than to M. javanica or M. incognita.     

Host Suitability of the Olive Cultivars Arbequina and Picual for Plant-Parasitic Nematodes

Host suitability of olive cultivars Arbequina and Picual to several plant-parasitic nematodes was studied under controlled conditions. Arbequina and Picual were not suitable hosts for the root-lesion nematodes Pratylenchus fallax, P. thornei, and Zygotylenchus guevarai. However, the ring nematode Mesocriconema xenoplax and the spiral nematodes Helicotylenchus digonicus and H. pseudorobustus reproduced on both olive cultivars. The potential of Meloidogyne arenaria race 2, M. incognita race 1, and M. javanica, as well as P. vulnus and P. penetrans to damage olive cultivars, was also assessed. Picual planting stocks infected by root-knot nematodes showed a distinct yellowing affecting the uppermost leaves, followed by a partial defoliation. Symptoms were more severe on M. arenaria and M. javanica-infected plants than on M. incognita-infected plants. Inoculation of plants with 15,000 eggs + second-stage juveniles/pot of these Meloidogyne spp. suppressed the main height of shoot and number of nodes of Arbequina, but not Picual. Infection by each of the two lesion nematodes (5,000 nematodes/pot) or by each of the three Meloidogyne spp. suppressed (P < 0.05) the main stem diameter of both cultivars. On Arbequina, the reproduction rate of Meloidogyne spp. was higher (P < 0.05) than that of Pratylenchus spp.; on Picual, Pratylenchus spp. reproduction was higher (P < 0.05) than that of Meloidogyne spp.     

The Surface Coat of Plant-Parasitic Nematodes: Chemical Composition,Origin, and Biological Role—A Review

Chemical composition, origin, and biological role of the surface coat (SC) of plant-parasitic nematodes are described and compared with those of animal-parasitic and free-living nematodes. The SC of the plant-parasitic nematodes is 5-30 nm thick and is characterized by a net negative charge. It consists, at least in part, of glycoproteins and proteins with various molecular weights, depending upon the nematode species. The lability of its components and the binding of human red blood cells to the surface of many tylenchid plant-parasitic nematodes, as well as the binding of several neoglycoproteins to the root-knot nematode Meloidogyne, suggest the presence of carbohydrate-recognition-domains for host plants and parasitic or predatory soil microorganisms (Pasteuria penetrans and Dactylaria spp., for example). These features may also assist in nematode adaptations to soil environments and to plant hosts with defense mechanisms that depend on reactions to nematode surfaces. Surface coat proteins can be species and race specific, a characteristic with promising diagnostic potential.     

Effects of Monoclonal Antibodies,Cationized Ferritin,and Other Organic Molecules on Adhesion of Pasteuria penetrans Endospores to Meloidogyne incognita

The incidence of adhesion of Pasteuria penetrans endospores to Meloidogyne incognita second-stage juveniles (J2) was studied after pretreatment of the latter with monoclonal antibodies (MAb), cationized ferritin, and other organic molecules in replicated trials. Monoclonal antibodies developed to a cuticular epitope of M. incognita second-stage juveniles gave significant reductions in attachment of P. penetrans endospores to treated nematodes. MAb bound to the entire length of J2 except for the area of the lateral field, where binding was restricted to the incisures. Since reductions in attachment with MAb treatment were modest, it is uncertain if these results implicated a specific surface protein as a factor that interacted in binding of the endospore to the nematode cuticle. Endospore attachment was decreased following treatment of the nematode with the detergents sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). Endospore attachment to live nematodes was significantly greater than attachment to dead nematodes. Attachment rates of three P. penetrans isolates to M. incognita race 3 varied between isolates. The effects of neuraminidase, pronase, pepsin, trypsin, lipase, and Na periodate on endospore attachment were inconsistent. The cationic dye alcian blue, which binds sulfate and carboxyl groups on acidic glycans, had no consistent effect on endospore attachment. The incidence of endospore attachment was significantly lower but modest, at best, for nematodes that were treated with cationized ferritin alone or cationized ferritin following monoclonal antibody. The lack of consistency or extreme reduction in most experiments suggests that attachment of P. penetrans spores to M. incognita is not specified by only one physico-chemical factor, but may involve a combination of at least two physico-chemical factors (including surface charge and movement of the J2). This points to a need for analysis of combined or factorial treatment effects.     

Transfer and Development of Pasteuria penetrans

Pasteuria penetrans isolate P-20 has been attributed as the cause of soil suppressiveness to peanut root-knot nematode in Florida. In this study, P. penetrans was transferred from a suppressive site to a new site and established by growing susceptible hosts to the peanut root-knot nematode during both summer and winter seasons. When two soil fumigants, 1,3-dichloropropene (1,3-D) and chloropicrin, were applied broadcast at the rate of 168 liters/ha and 263 kg/ha, respectively, the bacterium was not adversely affected by 1,3-D but was adversely affected by chloropicrin. In autumn 2005, after the harvest of the second peanut crop, the greatest number of J2 was recorded in the chloropicrin-treated plots, followed by the non-fumigated plots and 1,3-D-fumigated plots. The percentage J2 encumbered with endospores, endospores per J2 and percentage of P. penetrans-infected females were greatest in the non-fumigated plots, followed by 1,3-D- and chloropicrin-fumigated plots. This study demonstrates that P. penetrans can be transferred from a suppressive site to a new site and increased to suppressive densities against the peanut root-knot nematode.     

Parasitism of Helicotylenchus lobus by Pasteuria penetrans in Naturally Infested Soil

The population density of Helicotylenchus lobus and the percentage of the population with spores of Pasteuria penetrans were determined for 10 monthly intervals in naturally infested turf grass soil at Riverside, California. The percentage of nematodes with attached spores ranged from 40% to 67%. No relationship was found between nematode density and the percentage of nematodes with spores. The mean and maximum numbers of spores adhering per nematode with at least one spore ranged from 2 to 8 and 7 to 66, respectively. The mean number of spores per nematode (based on total number of H. lobus) was correlated with the percentage of nematodes with spores. Spores adhered to both adult and juvenile H. lobus. Between 9% and 32% of the nematodes with spores had been penetrated and infected by the bacterium. Many infected nematodes were dead, but mature spores were also observed within living adult and juvenile H. lobus that exhibited no apparent reduction in viability and motility. Spore and central endospore diameters of this P. penetrans isolate were larger than those reported for the type isolate from Meloidogyne incognita, but transmission and scanning electron microscopy did not reveal significant morphological differences between the two isolates. Spores of the isolate associated with H. lobus did not adhere to juveniles of M. incognita.     

Plant parasitic nematodes associated with ornamental plant Pseuderanthemum atropurpureum in Aligarh district of western Uttar Pradesh

A survey was conducted to determine the nematodes associated with the soil, substratum and roots of the ornamental plant, Pseuderanthemum atropurpureum, grown in the Aligarh district of western Uttar Pradesh, India. Ten genera of plant parasitic nematodes, viz. Meloidogyne spp., Helicotylenchus sp., Hemicriconemoides sp., Hoplolaimus sp., Xiphinema sp., Pratylenchus sp., Trichodorus sp., Tylenchorhynchus sp., Aphelenchoides sp. and Rotylenchus sp., were isolated from 345 soil samples collected from 15 different localities. Our study indicates that among the 10 genera of the plant parasitic nematodes, Meloidogyne spp. was widely spread in all the examined localities of Aligarh district, except at Khair. It was observed that the population density and diversity of nematodes in all the soil samples was not uniform. Meloidogyne spp. showed the highest absolute frequency and relative frequency followed by Helicotylenchus sp., Hemicriconemoides sp., Xiphinema sp., Hoplolaimus sp., Pratylenchus sp., Trichodorus sp., Tylenchorhynchus sp., Aphelenchoides sp. and Rotylenchus sp. Since no records are available in the literature related to the association of root-knot nematode in P. atropurpureum, it is worthy to note that this study may be considered as a new hope towards further study of the interaction between the root-knot nematode and ornamental plant P. atropurpureum if any.     

Estimating Incidence of Attachment of Pasteuria penetrans Endospores to Meloidogyne spp. with Tally Thresholds

Pasteuria penetrans has .been identified as an important biological control agent of root-knot nematodes. In this study the use of tally thresholds was evaluated for estimating P. penetrans endospore attachment to second-stage juveniles (J2) of Meloidogyne spp. A tally threshold (T) is defined as the maximum number of individuals in a sample unit that may be treated as absent based on binomial sampling. Three different data sets that originated from centrifugal bioassay, incubation bioassay, and field experiments were investigated. The data sets each contained 70, 33, and 111 estimates of the mean number of endospores attached per J2 (m), respectively. Empirical relationships between m and proportions of J2 with ≤T endospores attached (P_T) were developed using parameters from the linear regression of ln(m) on P_T (0 < P_T < 1): ln(m) = a + b P_T, T was set to 0, 1, 2, 3, 4, 5, 8, and 10 endospores/J2. The results indicated that the variances of linear equations tended to decrease with increasing T values for all three data sets. T values of 0, 1, 8, and 10 endospores/J2 for centrifugal bioassay and incubation bioassay, and of 0, 1, 2, and 3 endospores/J2 for field experiments were associated with an r² of >= 0.8. These T values were robust for estimating m from P_T, reducing the variability as well as the time and effort spent in estimating the mean number of endospores attached per J2.     

<Emphasis Type="Italic">CaMi</Emphasis>, a root-knot nematode resistance gene from hot pepper (<Emphasis Type="Italic">Capsium annuum</Emphasis> L.) confers nematode resistance in tomato

Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes. Rugang Chen and Hanxia Li are contributed equally to this work.     

Differential expression of root-knot nematode resistance genes in tomato and pepper: evidence with Meloidogyne incognita virulent and avirulent near-isogenic lineages

Reproduction of artificially selected near isogenic Meloidogyne incognita lineages virulent and avirulent against the Mi resistance gene of tomato was assessed on host and resistant lines and cultivars of pepper. Egg mass production following inoculation of individual potted seedlings with second-stage juveniles was studied in experiments conducted in controlled environment. Artificially selected Mi-virulent nematode populations were unable to develop on resistant pepper lines PM 217 and PM 687. This suggests that the genetic systems governing resistance to root-knot nematodes are differently expressed in tomato and pepper, in spite of the very close phylogenetic relationships and structural genomic homologies occurring between these two vegetable crops. Moreover, these artificially selected nematode populations were also found unable to develop on the susceptible pepper cultivars California Wonder and Doux Long des Landes, while their pathogenicity was not significantly affected on susceptible tomatoes. Due to the existence of naturally virulent Meloidogyne populations, these results enhance the need for a better understanding of the mechanisms involved, in order to develop new forms of management of plant resistance to root-knot nematodes.     

Compatibility Between Pasteuria penetrans Isolates and Meloidogyne Populations from Spain

Several populations of Pasteuria isolated from fields in Spain were compared with other Pasteuria populations, held in collections at the Institute de Recerca i Tecnologia, Agroalimentaries (IRTA), Cabrils or IACR-Rothamsted, for their ability to adhere to and infect root-knot nematodes ( Meloidogyne spp.) grown on host plants differing in their susceptibility to root-knot nematodes. The results showed a high level of variation in both the ability of a population of Pasteuria to adhere to a particular population of nematode and vice versa. In particular the isolates of Pasteuria originating from M. hapla retained a high level of specificity for the species from which they originated. The infection of the nematodes by the bacteria was generally low, even when nematodes were encumbered with relatively high levels of spores. It is suggested that prolonged storage (6 years) may reduce the ability of spores to infect nematodes independently of adhesion.     

Response of Resistant and Susceptible Bell Pepper (Capsicum annuum) to a Southern California Meloidogyne incognita Population from a Commercial Bell Pepper Field

To determine the presence and level of root-knot nematode (Meloidogyne spp.) infestation in Southern California bell pepper (Capsicum annuum) fields, soil and root samples were collected in April and May 2012 and analyzed for the presence of root-knot nematodes. The earlier samples were virtually free of root-knot nematodes, but the later samples all contained, sometimes very high numbers, of root-knot nematodes. Nematodes were all identified as M. incognita. A nematode population from one of these fields was multiplied in a greenhouse and used as inoculum for two repeated pot experiments with three susceptible and two resistant bell pepper varieties. Fruit yields of the resistant peppers were not affected by the nematodes, whereas yields of two of the three susceptible pepper cultivars decreased as a result of nematode inoculation. Nematode-induced root galling and nematode multiplication was low but different between the two resistant cultivars. Root galling and nematode reproduction was much higher on the three susceptible cultivars. One of these susceptible cultivars exhibited tolerance, as yields were not affected by the nematodes, but nematode multiplication was high. It is concluded that M. incognita is common in Southern California bell pepper production, and that resistant cultivars may provide a useful tool in a nonchemical management strategy.     

Temperature Effects on the Attachment of Pasteuria penetrans Endospores to Meloidogyne arenaria Race 1

Pasteuria penetrans is a gram positive bacterium that prevents Meloidogyne spp. from reproducing and diminishes their ability to penetrate roots. The attachment of the endospores to the cuticle of the nematodes is the first step in the life cycle of the bacterium and is essential for its reproduction. As a preliminary study to a field solarization test, the effects of temperature on the attachment of P. penetrans on Meloidogyne arenaria race 1 were investigated. Preexposing second-stage juveniles (J2) of M. arenaria to approximately 30 °C in water before exposing them to endospores increased their receptivity to endospore attachment when compared to treating J2 at 25 °C or 35 °C. In tests with soil, highest attachment occurred when J2 were incubated in soil infested with endospores and maintained at 20 °C to 30 °C for 4 days. Heating J2 in soil to sublethal temperatures (35 °C to 40 °C) decreased endospore attachment. Incubating P. penetrans endospores in soil at 30 °C to 70 °C for 5 hours a day over 10 days resulted in reductions of endospore attachment to nematodes as temperatures of incubation increased to 50 °C and higher.