Sewage sludge amendment and inoculation with plant-parasitic nematodes do not facilitate the internalization of Salmonella Typhimurium LT2 in lettuce plants.

Contamination of fruits and vegetables with Salmonella is a serious threat to human health. In order to prevent possible contaminations of fresh produce it is necessary to identify the contributing ecological factors. In this study we investigated whether the addition of sewage sludge or the presence of plant-parasitic nematodes foster the internalization of Salmonella enterica serovar Typhimurium LT2 into lettuce plants, posing a potential threat for human health. Greenhouse experiments were conducted to investigate whether the amendment of sewage sludge to soil or the presence of plant-parasitic nematodes Meloidogyne hapla or Pratylenchus crenatus promote the internalization of S. Typhimurium LT2 from soil into the edible part of lettuce plants. Unexpectedly, numbers of cultivable S. Typhimurium LT2 decreased faster in soil with sewage sludge than in control soil but not in root samples. Denaturing gradient gel electrophoresis analysis revealed shifts of the soil bacterial communities in response to sewage sludge amendment and time. Infection and proliferation of nematodes inside plant roots were observed but did not influence the number of cultivable S. Typhimurium LT2 in the root samples or in soil. S. Typhimurium LT2 was not detected in the leaf samples 21 and 49 days after inoculation. The results indicate that addition of sewage sludge, M. hapla or P. crenatus to soil inoculated with S. Typhimurium LT2 did not result in an improved survival in soil or internalization of lettuce plants.

Effector gene vap1 based DGGE fingerprinting to assess variation within and among Heterodera schachtii populations.

Populations of beet cyst nematodes Heterodera schachtii vary in aggressiveness and virulence toward sugar beet varieties, but also in traits like host range, or decline rate in the field. Diversity of their essential pathogenicity gene vap1 is shaped by diversifying selection and gene flow. The authors developed a technique to study inter-population variation and intra-population diversity and dynamics of H. schachtii based on the gene vap1. Degenerate primers were designed to amplify, clone, and sequence this gene from diverse species and populations of cyst nematodes. This resulted in a high diversity of sequences for H. schachtii, and allowed to design non-degenerated primers to amplify a fragment suitable for sequence dependent separation of gene variants in denaturing gradient gel electrophoresis (DGGE). The developed primers span a highly variable intron and part of a slightly variable exon. A marker comprised of the 14 mostly detected gene variants was established for gel-to-gel comparisons. For individual juveniles up to six gene variants were resolved and substantial variation within and among cysts was observed. A fast and easy DNA extraction procedure for 20 pooled cysts was established, which provided DGGE patterns with high similarity among replicate samples from field populations. Permutation tests on pairwise similarities within and among populations showed significant differences among vap1 patterns of field populations of H. schachtii. Similarly, gene diversity as expressed by the Shannon index was statistically different among field populations. In conclusion, the DGGE technique is a fast and - compared to sequencing approaches - inexpensive tool to compare populations of H. schachtii and link observed biological characteristics to genetic pattern.Populations of beet cyst nematodes Heterodera schachtii vary in aggressiveness and virulence toward sugar beet varieties, but also in traits like host range, or decline rate in the field. Diversity of their essential pathogenicity gene vap1 is shaped by diversifying selection and gene flow. The authors developed a technique to study inter-population variation and intra-population diversity and dynamics of H. schachtii based on the gene vap1. Degenerate primers were designed to amplify, clone, and sequence this gene from diverse species and populations of cyst nematodes. This resulted in a high diversity of sequences for H. schachtii, and allowed to design non-degenerated primers to amplify a fragment suitable for sequence dependent separation of gene variants in denaturing gradient gel electrophoresis (DGGE). The developed primers span a highly variable intron and part of a slightly variable exon. A marker comprised of the 14 mostly detected gene variants was established for gel-to-gel comparisons. For individual juveniles up to six gene variants were resolved and substantial variation within and among cysts was observed. A fast and easy DNA extraction procedure for 20 pooled cysts was established, which provided DGGE patterns with high similarity among replicate samples from field populations. Permutation tests on pairwise similarities within and among populations showed significant differences among vap1 patterns of field populations of H. schachtii. Similarly, gene diversity as expressed by the Shannon index was statistically different among field populations. In conclusion, the DGGE technique is a fast and ­ compared to sequencing approaches ­ inexpensive tool to compare populations of H. schachtii and link observed biological characteristics to genetic pattern.

Specific microbial attachment to root knot nematodes in suppressive soil.

Understanding the interactions of plant-parasitic nematodes with antagonistic soil microbes could provide opportunities for novel crop protection strategies. Three arable soils were investigated for their suppressiveness against the root knot nematode Meloidogyne hapla. For all three soils, M. hapla developed significantly fewer galls, egg masses, and eggs on tomato plants in unsterilized than in sterilized infested soil. Egg numbers were reduced by up to 93%. This suggested suppression by soil microbial communities. The soils significantly differed in the composition of microbial communities and in the suppressiveness to M. hapla. To identify microorganisms interacting with M. hapla in soil, second-stage juveniles (J2) baited in the test soil were cultivation independently analyzed for attached microbes. PCR-denaturing gradient gel electrophoresis of fungal ITS or 16S rRNA genes of bacteria and bacterial groups from nematode and soil samples was performed, and DNA sequences from J2-associated bands were determined. The fingerprints showed many species that were abundant on J2 but not in the surrounding soil, especially in fungal profiles. Fungi associated with J2 from all three soils were related to the genera Davidiella and Rhizophydium, while the genera Eurotium, Ganoderma, and Cylindrocarpon were specific for the most suppressive soil. Among the 20 highly abundant operational taxonomic units of bacteria specific for J2 in suppressive soil, six were closely related to infectious species such as Shigella spp., whereas the most abundant were Malikia spinosa and Rothia amarae, as determined by 16S rRNA amplicon pyrosequencing. In conclusion, a diverse microflora specifically adhered to J2 of M. hapla in soil and presumably affected female fecundity.

Identification of msp1 Gene Variants in Populations of Meloidogyne incognita Using PCR-DGGE.

Effectors of root-knot nematodes are essential for parasitism and prone to recognition by adapted variants of the host plants. This selective pressure initiates hypervariability of effector genes. Diversity of the gene variants within nematode populations might correlate with host preferences. In this study we developed a method to compare the distribution of variants of the effector gene msp1 among populations of Meloidogyne incognita. Primers were designed to amplify a 234-bp fragment of msp1. Sequencing of cloned PCR products revealed five msp1 variants from seven populations that were distinguishable in their reproduction on five host plants. A protocol for denaturing gradient gel electrophoresis (DGGE) was developed to separate these msp1 variants. DGGE for replicated pools of juveniles from the seven populations revealed ten variants of msp1. A correlation between the presence of a particular gene variant and the reproductive potential on particular hosts was not evident. Especially race 3 showed substantial variation within the population. DGGE fingerprints of msp1 tended to cluster the populations according to their reproduction rate on pepper. The developed method could be useful for analyzing population heterogeneity and epidemiology of M. incognita.

Host Status and Response Differences of Flat-Leaf and Curly-Leaf Parsley to Meloidogyne hapla, M. chitwoodi, M. fallax, and M. incognita Infestation.

Leaf parsley growth and productivity are often affected by pathogen infection. Root-knot nematodes of the genus Meloiogyne are common pathogens reported on leaf parsley. The response of leaf parsley to Meloidogyne species in tropical and subtropical regions is quite known, while in temperate regions, comparable information is still scarce. In this study, we evaluated the host status and response of three flat-leaf (Laica, Laura, Gigante d'Italia) and three curly-leaf (Grüne Perle, Orfeo, Sombre) parsley cultivars to Meloidogyne species from temperate regions, i.e., M. hapla, M. chitwoodi, and M. fallax, as well as to the southern root-knot nematode M. incognita. Evaluation was based on measuring plant biomass and nematode reproduction nine weeks after nematode inoculation. Our results showed that all four Meloidogyne species did not cause the reduction in leaf parsley growth under the given experimental conditions. Regarding the host status of leaf parsley cultivars for Meloidogyne, results were variable. All six parsley cultivars were found to be good hosts for M. hapla. Regarding M. chitwoodi, the host status could not be clarified properly; however, each cultivar allowed nematode reproduction at least in one experiment. For M. fallax, flat-leaf parsley turned out to be less susceptible than curly-leaf parsley; and for M. incognita, Orfeo, Laura, and Laica were classified as good hosts, Grüne Perle and Sombre as poor hosts, and Gigante d'Italia as a non-host. Amongst all tested cultivars, Gigante d'Italia was found to be the least susceptible cultivar due to its poor host status for M. chitwoodi and non-host status for M. fallax and M. incognita. Infection with M. hapla, M. chitwoodi, and M. incognita, but not with M. fallax, resulted in distinct gall formation on the roots of all six leaf parsley cultivars.

Morphometric and Molecular Diversity among Seven European Isolates of Pratylenchus penetrans.

Pratylenchus penetrans is an economically important root-lesion nematode species that affects agronomic and ornamental plants. Understanding its diversity is of paramount importance to develop effective control and management strategies. This study aimed to characterize the morphological and genetic diversity among seven European isolates. An isolate from the USA was included in the molecular analyses for comparative purposes. Morphometrics of the European P. penetrans isolates generally were within the range of the original descriptions for this species. However, multiple morphometric characteristics, including body length, maximum body width, tail length and length of the post-vulval uterine sac showed discrepancies when compared to other populations. Nucleotide sequence-based analyses revealed a high level of intraspecific diversity among the isolates. We observed no correlation between D2-D3 rDNA- and COXI-based phylogenetic similarities and geographic origin. Our phylogenetic analyses including selected GenBank sequences also suggest that the controversy surrounding the distinction between P. penetrans and P. fallax remains.

New Insights on the Role of Allyl Isothiocyanate in Controlling the Root Knot Nematode Meloidogyne hapla.

Biofumigation, although a well-known method, is still controversially debated as a management strategy for plant-parasitic nematodes (PPN). Its controlling effect is attributed to the production of isothiocyanates (ITCs) following the action of myrosinase on glucosinolates (GSLs). Different ITCs are formed from different GSLs, depending on the plant species. To better understand the potential of ITCs, eight cultivars from three Brassicaceae species were investigated as biofumigation crops to control the root knot nematode Meloidogyne hapla. Since results were inconsistent, the nematicidal effect of selected ITCs were further evaluated in vitro. Based on its nematicidal potential, allyl ITC (AITC) was specifically investigated under different soil:sand compositions. A significantly lower nematicidal activity was observed in soil compared to sand. AITC was also evaluated as an additive to the biofumigation in a greenhouse trial. Its supplementation to the biofumigation process with Brassica juncea cv. Terrafit controlled M. hapla, while no control was observed using Raphanus sativus cv. Defender. Thus, the success of biofumigation seems to be strongly dependent on the soil characteristics and the ITC produced during the biofumigation process. Therefore, the supplementation of AITC in combination with the right cover crop can improve the biofumigation process to control M. hapla.

Symbiosis of soybean with nitrogen fixing bacteria affected by root lesion nematodes in a density-dependent manner.

Early maturing varieties of soybean have a high yield potential in Europe, where the main biotic threat to soybean cultivation are root lesion nematodes (Pratylenchus spp.). Nitrogen fixation in root nodules by highly efficient inoculants of Bradyrhizobium japonicum is an incentive to grow soybean in low-input rotation systems. We investigated density-dependent effects of Pratylenchus penetrans on nitrogen fixation by co-inoculated B. japonicum. Less than 130 inoculated nematodes affected the number and weight of nodules, the density of viable bacteroids in nodules, and nitrogen fixation measured as concentration of ureides in leaves. With more inoculated nematodes, the percentage that invaded the roots increased, and adverse effects on the symbiosis accelerated, leading to non-functional nodules at 4,000 and more nematodes. When P. penetrans invaded roots that had fully established nodules, growth of nodules, density of bacteroids, and nitrogen fixation were affected but not the number of nodules. In contrast, nodulation of already infested roots resulted in a high number of small nodules with decreased densities of bacteroids and nitrogen fixation. P. penetrans invaded and damaged the nodules locally, but they also significantly affected the nodule symbiosis by a plant-mediated mechanism, as shown in an experiment with split-root systems.

Bacteria isolated from the cuticle of plant-parasitic nematodes attached to and antagonized the root-knot nematode Meloidogyne hapla.

Plant-parasitic nematodes are associated with specifically attached soil bacteria. To investigate these bacteria, we employed culture-dependent methods to isolate a representative set of strains from the cuticle of the infective stage (J2) of the root-knot nematode Meloidogyne hapla in different soils. The bacteria with the highest affinity to attach to J2 belonged to the genera Microbacterium, Sphingopyxis, Brevundimonas, Acinetobacter, and Micrococcus as revealed by 16S rRNA gene sequencing. Dynamics of the attachment of two strains showed fast adhesion in less than two hours, and interspecific competition for attachment sites. Isolates from the cuticle of M. hapla J2 attached to the lesion nematode Pratylenchus penetrans, and vice versa, suggesting similar attachment sites on both species. Removal of the surface coat by treatment of J2 with the cationic detergent CTAB reduced bacterial attachment, but did not prevent it. Some of the best attaching bacteria impaired M. hapla performance in vitro by significantly affecting J2 mortality, J2 motility and egg hatch. Most of the tested bacterial attachers significantly reduced the invasion of J2 into tomato roots, suggesting their beneficial role in soil suppressiveness against M. hapla.

Rhizosphere Microbiomes Modulated by Pre-crops Assisted Plants in Defense Against Plant-Parasitic Nematodes.

Plant-parasitic nematodes cause considerable damage to crop plants. The rhizosphere microbiome can affect invasion and reproductive success of plant-parasitic nematodes, thus affecting plant damage. In this study, we investigated how the transplanted rhizosphere microbiome from different crops affect plant-parasitic nematodes on soybean or tomato, and whether the plant's own microbiome from the rhizosphere protects it better than the microbiome from fallow soil. Soybean plants growing in sterilized substrate were inoculated with the microbiome extracted from the rhizosphere of soybean, maize, or tomato. Controls were inoculated with extracts from bulk soil, or not inoculated. After the microbiome was established, the root lesion nematode Pratylenchus penetrans was added. Root invasion of P. penetrans was significantly reduced on soybean plants inoculated with the microbiome from maize or soybean compared to tomato or bulk soil, or the uninoculated control. In the analogous experiment with tomato plants inoculated with either P. penetrans or the root knot nematode Meloidogyne incognita, the rhizosphere microbiomes of maize and tomato reduced root invasion by P. penetrans and M. incognita compared to microbiomes from soybean or bulk soil. Reproduction of M. incognita on tomato followed the same trend, and it was best suppressed by the tomato rhizosphere microbiome. In split-root experiments with soybean and tomato plants, a systemic effect of the inoculated rhizosphere microbiomes on root invasion of P. penetrans was shown. Furthermore, some transplanted microbiomes slightly enhanced plant growth compared to uninoculated plants. The microbiomes from maize rhizosphere and bulk soil increased the fresh weights of roots and shoots of soybean plants, and microbiomes from soybean rhizosphere and bulk soil increased the fresh weights of roots and shoots of tomato plants. Nematode invasion did not affect plant growth in these short-term experiments. In conclusion, this study highlights the importance of the rhizosphere microbiome in protecting crops against plant-parasitic nematodes. An effect of pre-crops on the rhizosphere microbiome might be harnessed to enhance the resistance of crops towards plant-parasitic nematodes. However, nematode-suppressive effects of a particular microbiome may not necessarily coincide with improvement of plant growth in the absence of plant-parasitic nematodes.

Low amounts of herbivory by root-knot nematodes affect microbial community dynamics and carbon allocation in the rhizosphere.

Increased carbon translocation to the rhizosphere via 'leakage' induced by low amounts of plant parasitic nematodes can foster microorganisms. The effects of the root-knot nematode Meloidogyne incognita on microbial biomass (C(mic)) and community structure (phospholipid fatty acids) in the rhizosphere of barley were studied. Inoculation densities of 2000, 4000, and 8000 nematodes were well below the threshold level for plant damage. A (13)CO(2) pulse-labelling was performed to assess the distribution of assimilated (13)C in the rhizosphere. Infection with M. incognita increased the carbon concentration in shoots, and enhanced root biomass slightly. The presence of nematodes did not affect microbial biomass, but significantly changed the allocation of the recent photosynthate. Less plant carbon was sequestered by microorganisms with increasing nematode abundance. Microbial community structure was distinctly altered in the early stages of the plant-nematode interactions. Both, bacteria and fungi, showed a positive response with 2000, and a negative one with 4000 and 8000 M. incognita added. The results suggest that low-level root herbivory still imposes a considerable carbon demand, and that proliferation of microorganisms due to increased rhizodeposition may be short-termed. The carbon flow to rhizosphere microbial communities is likely dependent on the specific nematode-plant association and the developmental stage of the nematode in the host.

Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi.

Differences between endophytic and ectophytic bacterial communities with stress on antagonistic bacteria, were studied by comparing the composition of communities isolated from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato plants using a multiphasic approach. Terminal restriction fragment length polymorphism analysis of 16S rDNA of the bacterial communities revealed discrete microenvironment-specific patterns. To measure the antagonistic potential of potato-associated bacteria, a total of 2648 bacteria were screened by dual testing of antagonism to the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. Composition and diversity of bacterial antagonists were mainly specific for each microenvironment. The rhizosphere and endorhiza were the main reservoirs for antagonistic bacteria and showed the highest similarity in their colonisation by antagonists. The most prominent species of all microenvironments was Pseudomonas putida, and rep-PCR with BOX primers showed that these isolates showed microenvironment-specific DNA fingerprints. P. putida isolates from the rhizosphere and endorhiza gave nearly identical fingerprints confirming the high similarity of bacterial populations. The phlD gene, involved in the production of the antibiotic 2,4-diacetyl-phloroglucinol, was found only among Pseudomonas isolates from the rhizosphere and endorhiza. Evaluation of the bacterial isolates for biocontrol potential based on fungal antagonism and physiological characteristics resulted in the selection of five promising isolates from each microenvironment. The most effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.

Bacterial antagonists of fungal pathogens also control root-knot nematodes by induced systemic resistance of tomato plants.

The potential of bacterial antagonists of fungal pathogens to control the root-knot nematode Meloidogyne incognita was investigated under greenhouse conditions. Treatment of tomato seeds with several strains significantly reduced the numbers of galls and egg masses compared with the untreated control. Best performed Bacillus subtilis isolates Sb4-23, Mc5-Re2, and Mc2-Re2, which were further studied for their mode of action with regard to direct effects by bacterial metabolites or repellents, and plant mediated effects. Drenching of soil with culture supernatants significantly reduced the number of egg masses produced by M. incognita on tomato by up to 62% compared to the control without culture supernatant. Repellence of juveniles by the antagonists was shown in a linked twin-pot set-up, where a majority of juveniles penetrated roots on the side without inoculated antagonists. All tested biocontrol strains induced systemic resistance against M. incognita in tomato, as revealed in a split-root system where the bacteria and the nematodes were inoculated at spatially separated roots of the same plant. This reduced the production of egg masses by up to 51%, while inoculation of bacteria and nematodes in the same pot had only a minor additive effect on suppression of M. incognita compared to induced systemic resistance alone. Therefore, the plant mediated effect was the major reason for antagonism rather than direct mechanisms. In conclusion, the bacteria known for their antagonistic potential against fungal pathogens also suppressed M. incognita. Such "multi-purpose" bacteria might provide new options for control strategies, especially with respect to nematode-fungus disease complexes that cause synergistic yield losses.

Bacillus and Streptomyces were selected as broad-spectrum antagonists against soilborne pathogens from arid areas in Egypt.

Plant protection via disease-suppressive bacteria in desert farming requires specific biological control agents (BCAs) adapted to the unique arid conditions. We performed an ecological study of below-ground communities in desert farm soil and untreated desert soil, and based on these findings, selected antagonists were hierarchically evaluated. In contrast to the highly specific 16S rRNA fingerprints of bacterial communities in soil and cultivated medicinal plants, internal transcribed spacer profiles of fungal communities were less discriminative and mainly characterised by potential pathogens. Therefore, we focused on in vitro bacterial antagonists against pathogenic fungi. Based on the antifungal potential and genomic diversity, 45 unique strains were selected and characterised in detail. Bacillus/Paenibacillus were most frequently identified from agricultural soil, but antagonists from the surrounding desert soil mainly belonged to Streptomyces. All strains produced antibiotics against the nematode Meloidogyne incognita, and one-third showed additional activity against the bacterial pathogen Ralstonia solanacearum. Altogether, 13 broad-spectrum antagonists with antibacterial, antifungal and nematicidal activity were found. They belong to seven different bacterial species of the genera Bacillus and Streptomyces. These Gram-positive, spore-forming bacteria are promising drought-resistant BCAs and a potential source for antibiotics. Their rhizosphere competence was shown by fluorescence in situ hybridisation combined with laser scanning microscopy.

Effects of pyrrolizidine alkaloids on the performance of plant-parasitic and free-living nematodes.

BACKGROUND: Chemical nematicides such as methyl bromide have for decades played a significant role in the management of plant-parasitic nematodes. Their application is problematic because of negative environmental impacts, and therefore methyl bromide was phased out in Europe in 2005. A possible alternative to synthetically derived nematicides is seen in the use of plants and/or their secondary metabolites. These plants could either be used as nematicidal green manure or as a source for nematicidal extracts. This study aimed to evaluate the effects of 1,2-dehydropyrrolizidine alkaloids (PAs), a group of secondary plant metabolites found in hundreds of plant species throughout the world, on the performance of plant-parasitic and free-living nematodes. RESULTS: PAs induced nematicidal, ovicidal and repellent effects on different plant-parasitic and free-living nematodes. There was no conclusive ranking in toxicity for the different structural types of PAs tested. However, the effects were often more pronounced for the tertiary than for the oxidised form of PAs. Further, large differences were observed in the susceptibility of different nematode species to PAs. CONCLUSIONS: PAs do affect several performance parameters and developmental stages of nematodes. Therefore, PA-producing plants such as species of Crotalaria, Ageratum or Senecio might be promising candidates for nematode management strategies. [Correction made here after initial online publication].

Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood.

To study the effect of microenvironments on potato-associated bacteria, the abundance and diversity of bacteria isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field grown potato was analyzed. Culturable bacteria were obtained after plating on R2A medium. The endophytic populations averaged 10(3) and 10(5) CFU/g (fresh wt.) for the endosphere and endorhiza. respectively, which were lower than those for the ectophytic microenvironments, with 10(5) and 10(7) CFU/g (fresh wt.) for the phyllosphere and rhizosphere, respectively. The composition and richness of bacterial species was microenvironment-dependent. The occurrence and diversity of potato-associated bacteria was additionally monitored by a cultivation-independent approach using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of microenvironment-specific communities. In an approach to measure the antagonistic potential of potato-associated bacteria, a total of 440 bacteria was screened by dual testing for in vitro antagonism towards the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. The proportion of isolates with antagonistic activity was highest for the rhizosphere (10%), followed by the endorhiza (9%), phyllosphere (6%), and endosphere (5%). All 33 fungal antagonists were characterized by testing their in vitro antagonistic mechanisms, including their glucanolytic, chitinolytic, pectinolytic, cellulolytic, and proteolytic activity, and by their BOX-PCR fingerprints. In addition, they were screened for their biocontrol activity against Meloidogyne incognita. Overall, nine isolates belonging to Pseudomonas and Streptomyces species were found to control both fungal pathogens and M. incognita and were therefore considered as promising biological control agents.