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Isolation and molecular characterization of five entomopathogenic nematode species and their bacterial symbionts from eastern Australia

• Published: 17 August 2021
• Volume 67 , pages 63–74, ( 2022 )

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• Sitaram Aryal   ORCID: orcid.org/0000-0002-4265-7663 1 ,
• Uffe N. Nielsen   ORCID: orcid.org/0000-0003-2400-7453 1 ,
• Nanette H. Sumaya   ORCID: orcid.org/0000-0002-2645-1120 2 ,
• Stefano De Faveri   ORCID: orcid.org/0000-0001-5360-5570 3 ,
• Craig Wilson 4 &
• Markus Riegler   ORCID: orcid.org/0000-0001-7363-431X 1  

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Entomopathogenic nematodes (EPNs) are used in biological control of pest insects but their potential may be limited by strain availability from different bioregions and effectiveness against specific pests. Here, we isolated and characterized EPNs and their symbiotic bacteria from Australia where their diversity is scarcely known. We collected 198 soil samples from citrus orchards, grasslands and forests across temperate, subtropical and tropical eastern Australia. EPNs were isolated by baiting with mealworm, greater wax moth and Queensland fruit fly, The Australia’s most significant horticultural pest. We obtained 36 isolates which, according to DNA sequence analyses, represented five species, Heterorhabditis bacteriophora , Heterorhabditis indica , Heterorhabditis marelatus , Heterorhabditis zealandica and Steinernema feltiae , including the first report of H. marelatus from Australia, and H. indica and H. zealandica from New South Wales. Thirty-five isolates were baited with mealworm, one with fruit fly, and none with wax moth. Heterorhabditis marelatus was recovered from forests, H. bacteriophora from citrus orchards, S. feltiae from citrus orchards and grasslands, H. indica and H. zealandica from all three habitats. According to bacterial DNA analyses, Photorhabdus heterorhabditis occurred in H. zealandica and a reference strain of H. bacteriophora , Photorhabdus laumondii in H. bacteriophora and H. marelatus , Photorhabdus tasmaniensis in H. indica and H. bacteriophora , and Photorhabdus namnaonensis in H. zealandica . Unexpectedly, Pseudomonas protegens and Delftia acidovorans were found in S. feltiae while its expected symbiont Xenorhabdus remained undetected, possibly due to our approach. The newly isolated EPNs should be tested as biological control agents against pest insects.

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Data availability.

All sequence data have been deposited in GenBank (NCBI). All other data are contained within the manuscript and supplementary material.

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Acknowledgements

We thank Geraldine Tilden for technical support with fruit fly rearing, Alexander Robertson, Giles Ross, Alihan Katlav and Kylie Baker for help with field sampling, Michael Duncan for supply of wax moths and bees wax, and Roy Akhurst and Ian Broughton for advice.

This research was supported by the Australian Research Council Industrial Transformation Training Centre (ARC-ITTC) Fruit Fly Biosecurity Innovation (IC150100026), with a PhD scholarship to SA, and the Department of Agriculture, Water and the Environment’s Strengthening Australia’s Fruit Fly System Research Program; project: A national biocontrol program to manage pest fruit flies in Australia (4-EKSH327).

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Hawkesbury Institute of the Environment, Western Sydney University, Penrith, NSW, 2751, Australia

Sitaram Aryal, Uffe N. Nielsen & Markus Riegler

Department of Biological Sciences, College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology, Iligan City, Philippines

Nanette H. Sumaya

Department of Agriculture and Fisheries (DAF), Mareeba, QLD, Australia

Stefano De Faveri

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Contributions

SA, MR and UNN conceptualized and designed the experimental work. SA and MR collected material with support of SDF. CW provided additional material. SA performed the experiments, collected and analyzed the data, under guidance of MR and UNN, and advice of NHS and CW. MR was responsible for research funding. SA wrote the manuscript together with MR and UNN and with input of all other authors. All authors agree with the submission of the manuscript.

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Correspondence to Sitaram Aryal or Markus Riegler .

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The authors do not have a conflict of interest, except that CW works at Ecogrow, a company that supplied three EPN isolates which were characterised as part of this study.

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Aryal, S., Nielsen, U.N., Sumaya, N.H. et al. Isolation and molecular characterization of five entomopathogenic nematode species and their bacterial symbionts from eastern Australia. BioControl 67 , 63–74 (2022). https://doi.org/10.1007/s10526-021-10105-7

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Received : 23 March 2021

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Issue Date : February 2022

DOI : https://doi.org/10.1007/s10526-021-10105-7

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• Published: 13 June 2019

Improved phylogenomic sampling of free-living nematodes enhances resolution of higher-level nematode phylogeny

• Ashleigh B. Smythe 1 ,
• Oleksandr Holovachov 2 &
• Kevin M. Kocot   ORCID: orcid.org/0000-0002-8673-2688 3  

BMC Evolutionary Biology volume  19 , Article number:  121 ( 2019 ) Cite this article

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Nematodes are among the most diverse and abundant metazoans on Earth, but research on them has been biased toward parasitic taxa and model organisms. Free-living nematodes, particularly from the clades Enoplia and Dorylaimia, have been underrepresented in genome-scale phylogenetic analyses to date, leading to poor resolution of deep relationships within the phylum.

We supplemented publicly available data by sequencing transcriptomes of nine free-living nematodes and two important outgroups and conducted a phylum-wide phylogenomic analysis including a total of 108 nematodes. Analysis of a dataset generated using a conservative orthology inference strategy resulted in a matrix with a high proportion of missing data and moderate to weak support for branching within and placement of Enoplia. A less conservative orthology inference approach recovered more genes and resulted in higher support for the deepest splits within Nematoda, recovering Enoplia as the sister taxon to the rest of Nematoda. Relationships within major clades were similar to those found in previously published studies based on 18S rDNA.

Conclusions

Expanded transcriptome sequencing of free-living nematodes has contributed to better resolution among deep nematode lineages, though the dataset is still strongly biased toward parasites. Inclusion of more free-living nematodes in future phylogenomic analyses will allow a clearer understanding of many interesting aspects of nematode evolution, such as morphological and molecular adaptations to parasitism and whether nematodes originated in a marine or terrestrial environment.

Nematodes are ubiquitous and diverse metazoans that are found free-living in nearly every terrestrial and aquatic habitat and parasitizing most animals and plants. Fewer than 30,000 species have been described, but the actual diversity of the phylum may be closer to 1 million species [ 1 ]. Despite estimates that at least half of all nematodes are free-living [ 1 , 2 ], most research has focused on parasitic nematodes of medical and agricultural importance. Particularly neglected are the free-living marine nematodes, with only around 6900 species described [ 3 ] and no genomes published to date [ 4 ]. Of significance, free-living nematodes are generally the most abundant and diverse metazoans of marine sediments [ 5 , 6 , 7 , 8 ] where they are important as decomposers, predators, food for higher trophic levels [ 9 ], and as bioindicators for climate change and ecological disturbance [ 10 , 11 , 12 ].

Despite the importance of nematodes as free-living animals and as parasites of humans, livestock, and crops, and despite more than a century of intensive research, certain aspects of their origin and early evolution, such as the branching order near the root of Nematoda, are not yet fully understood [ 13 , 14 ]. Nematode evolutionary history is particularly interesting because of the diversity of niches they occupy – ranging from the blood and tissues of vertebrate and invertebrate animals, unicellular eukaryotes, all parts of plants, virtually every terrestrial habitat, and all aquatic environments including deep-sea hydrothermal vent communities – is unrivaled in Metazoa [ 15 , 16 , 17 , 18 ]. Thus, resolving nematode phylogeny, especially the branching order close to the root of the nematode tree, will not only improve our understanding of the origin of economically important groups, but will provide a phylogenetic framework for understanding the underlying key characters (e.g., genomic modifications) corresponding to different nematode lifestyles, advancing all aspects of nematology, from basic evolutionary biology to pathogen control and drug development [ 19 ].

Morphology-based hypotheses of higher-level nematode relationships (reviewed by [ 17 , 20 , 21 , 22 ]) placed emphasis on the presence or absence of a lateral canal excretory system and a number of esophageal features [ 23 , 24 ]. These characters were interpreted as evidence of two major lineages: a primarily terrestrial (but also including many plant and animal parasites) grouping called Secernentea, and a primarily aquatic grouping called Adenophorea. Subsequent morphological investigations by Andrássy [ 25 ] and Malakhov [ 26 ] distinguished three main lineages, elevating the primarily aquatic Enoplia and Chromadoria out of Adenophorea and re-classifying most Secernentea as Rhabditia.

The first molecular phylogenetic hypothesis for Nematoda used 18S rDNA [ 27 ] and differed substantially from previous morphology-based hypotheses of nematode phylogeny (e.g. [ 24 ]). This and subsequent analyses based on 18S have led to the recognition of three major lineages of nematodes: Dorylaimia (Clade I), Enoplia (Clade II), and Chromadoria, which consists of Spirurina (Clade III), Tylenchina (Clade IV), Rhabditina (Clade V), Plectida, Araeolaimida, Monhysterida, Desmodorida, and Chromadorida [ 17 , 21 , 22 , 27 , 28 , 29 ]. Dorylaimia includes many free-living soil nematodes and plant parasites, but also vertebrate parasites such as Trichinella, whipworms, and Dioctophyme . Enoplia primarily consists of free-living aquatic nematodes, but also several lineages of soil nematodes and virus-transmitting plant pests (such as stubby root nematodes). Chromadoria includes a wide diversity of free-living aquatic nematodes but also familiar animal parasites (e.g. Ascaris , hookworms, and Dirofilaria ), plant parasites (e.g. cyst and root knot nematodes), and the model organism Caenorhabditis elegans .

Enoplia has generally been thought to represent the sister group to all remaining nematodes [ 7 , 30 ] because of the presence of presumably ancestral developmental features, which are common in other animal phyla but not seen in other lineages of nematodes thus far investigated. These include indeterminate development [ 31 , 32 , 33 ] and retention of the nuclear envelope in mature spermatozoa (other nematodes investigated to date have determinate development and spermatozoa that lose the nuclear envelope upon maturation [ 34 ]). As other metazoan lineages are thought to have marine origins, nematodes have traditionally been assumed to have evolved in the marine environment [ 24 , 26 , 35 ]. Thus the primarily marine habits of Enoplia, combined with their presumed ancestral developmental features, have led to them being viewed as the earliest-branching nematode lineage [ 7 , 30 ]. On the other hand, De Ley and Blaxter [ 22 ] suggested the possibility of a terrestrial origin of nematodes with the sister group to all other nematodes being the taxon least represented in the marine environment, Dorylaimia. Ribosomal DNA-based studies have been unable to resolve the branching order among these deepest branches within Nematoda. Even studies focused on improved representation of diverse marine free-living nematodes [ 7 , 36 ] failed to find resolution at the base of the nematode tree, suggesting that additional molecular markers are needed to resolve deep nematode phylogeny.

Recently, phylogenomic studies employing dozens to hundreds of nuclear protein-coding genes have addressed questions of nematode evolution, but taxon sampling in these studies has largely built on publicly available genome and transcriptome datasets [ 4 , 37 , 38 , 39 , 40 , 41 ]. Until now, phylogenomic analyses of Nematoda have focused on parasitic taxa and model Caenorhabditis spp., with little or no representation of other free-living nematodes. For example, Blaxter and Koutsovoulos [ 40 ] and Koutsovoulos [ 41 ] curated the largest phylogenomic datasets for Nematoda to date but included only a single member of Enoplia in their studies. The latest comparative phylogenomic study focusing on parasitic worms included a handful of free-living nematodes (mostly model organisms), but no representatives of Enoplia or early branching Chromadoria [ 42 ]. Likewise, phylogenetic analyses based on mitochondrial genomes have never included representatives of Enoplia [ 43 , 44 , 45 , 46 ] because the mitochondrial genome has not yet been sequenced for any member of this clade.

Here, we have assembled the largest and most diverse phylogenomic dataset for Nematoda to date with expanded transcriptome representation for previously undersampled free-living nematode taxa. Leveraging this dataset, we re-examine relationships among early-branching clades and provide a robustly resolved and expanded phylogenetic framework for Nematoda.

Publicly available nematode and outgroup genomes and transcriptomes were supplemented with new transcriptomes from nine free-living nematodes, one nematomorph, and one kinorhynch for a total of 131 taxa sampled (Table  1 , Additional file  2 : Tables S1-S2). Building on an established phylogenomic data processing pipeline [ 47 ], we assembled two datasets using two different sequence selection strategies (see Methods ). The first strategy used a strict orthology inference approach that refines initial orthology inference made by HaMStR [ 48 ] with PhyloTreePruner [ 49 ]. This strategy resulted in a dataset with 931 genes totalling 298,009 amino acids in length with 84.67% missing data. The second strategy employed SCaFoS [ 50 ] to select the best sequence for each taxon in the HaMStR output. The SCaFoS strategy resulted in a dataset with 1025 genes totalling 321,951 amino acids in length with 35.01% missing data.

Our results based on the matrix assembled with the more conservative PhyloTreePruner orthology inference strategy but with a higher proportion of missing data (Fig.  1 ) strongly support nematode monophyly (IQ-TREE / RAxML bootstrap support, bs = 100%/100%), and subsequent branching, with Enoplia being monophyletic (as previously recovered [ 7 , 51 ]), and the sister clade to Dorylaimia and Chromadoria. Enoplida+Triplonchida was moderately supported (bs = 88%/74%). However, Enoplida was paraphyletic with respect to Triplonchida, a single representative of which, Tobrilus sp., was included as an ingroup. Dorylaimia and Chromadoria were recovered as sister taxa with strong support in the IQ-TREE analysis (bs = 99%) and moderate support in the RAxML analysis (bs = 80%).

Phylogeny of Nematoda based on the IQ-TREE maximum likelihood analysis of the PhyloTreePruner dataset. “Classification” bar on the left side serves as a scale and represents the relative known taxonomic diversity of different taxa within Nematoda: the height of each colored bar is proportional to a number of known species (also given in the brackets after each taxon name), with the height of the entire multicolored background rectangle equal to 100% of known nematode diversity. IQ-TREE / RAxML bootstrap support values < 100% are shown. “Habitat” describes the lifestyle for each analysed species, such as animal parasitic (animal par.), plant parasitic (plant par.), entomopathogenic or entomoparasitic (entomop.), free-living freshwater (freshwater), terrestrial (terrestrial) and marine (marine). Newly generated transcriptomes are marked with an asterisk

Dorylaimia was strongly supported (bs = 100%/96%). This clade was primarily represented by members of the animal parasitic Trichinellida ( Trichinella and Trichuris ), which was also strongly supported as monophyletic (bs =100%/100%). Dorylaimida, which was represented by the virus-transmitting plant pests Longidorus elongatus and Xiphinema index , was also strongly supported as monophyletic (bs =100%/100%). Monophyly could not be tested for the remaining three orders represented by just one taxon each: Mononchida (represented by Prionchulus punctatus ), Mermithida (represented by Romanomermis culicivorax ), and Dioctophymatida (represented by Soboliphyme baturini ). Mermithida was recovered as the sister to Mononchida with maximal support.

Chromadoria was strongly supported (bs =100%/100%) with the sole representative of Chromadorida ( Euchromadora sp.) sister to a well-supported (bs = 99%/82%) clade of all other Chromadoria with Odontophora sp., the single representative of Araeolaimida, at the base. The two sampled representatives of Plectida ( Plectus sambesii and Anaplectus granulosus ) were recovered in a clade (bs =100%/100%) sister to Rhabditida. Rhabditida includes most described species of Chromadoria, and also most of the currently available transcriptomes and genomes. It received maximal support as did its three subclades: Spirurina, Tylenchina, and Rhabditina. Relationships within the major clades of Rhabditida were also consistently strongly supported. All genera for which monophyly was testable (i.e., those with more than one representative available for study), were recovered monophyletic, with the exception of Heterorhabditis . Heterorhabditis bacteriophora was strongly supported as sister to a clade composed of Trichostrongylidae and Ancylostomatidae within Rhabditina (as expected), while a species identified as H. indica was strongly supported as the sister taxon of Globodera spp. in Tylenchina.

Examination of the Heterorhabditis indica dataset [ 52 ] revealed that this organism was incorrectly identified or mislabelled – partial sequences of the nuclear ribosomal operon mined from the H. indica transcriptome assembly show high similarity to reference sequences from various species of the genera Heterodera and Globodera (Hoplolaimidae, Tylenchina), and not Heterorhabditis (Heterorhabditidae, Rhabditina). This is further confirmed by the results of our tree-based taxonomy assignment using the 18S rDNA gene fragment (Additional file  1 : Figure S1). Unfortunately, these partial sequences mined from the transcriptome of H. indica are relatively short, one with only 588 bases of the 5′ end of 18S rDNA and the other with just 863 bases of the 5′ end of 28S rDNA. They do not contain enough phylogenetically informative sites to ensure species-level identification.

Because of the high amount of missing data (84.67%) in the dataset assembled using PhyloTreePruner, we also used a less conservative orthology inference approach that did not employ an additional tree-based orthology confirmation after initial HaMStR orthology inference. This resulted in a larger and much more complete dataset with 1026 genes totalling 321,951 amino acids in length with 64.99% matrix completeness. Analysis of this SCaFoS-based dataset resulted in a nearly identical branching order as that of the PhyloTreePruner-based dataset (Fig.  2 ). Whereas support for Enoplia was weak in the analysis of the PhyloTreePruner-based dataset, analysis of this dataset recovered Enoplia monophyletic and sister to the rest of Nematoda with maximal support. Tobrilus sp. (Triplonchida) was recovered sister to Enoplida with maximal support and Bathylaimus sp. was recovered sister to all other Enoplida with maximal support, which is in agreement with 18S rDNA-based analyses by van Megen et al. [ 53 ], Bik et al. [ 7 ], and Smythe [ 51 ]. Relationships within Dorylaimia were strongly supported and identical to the results based on the PhyloTreePruner dataset with the exception of relationships among Trichinella nativa , T. britovi , and T. murrelli. Likewise, relationships within Chromadoria were nearly identical; the one difference was placement of Oscheius tipulae , which was recovered sister to Rhabditomorpha sensu De Ley & Blaxter, 2004 [ 22 ] in the analysis of the PhyloTreePruner dataset and sister to Strongyloidea sensu De Ley & Blaxter, 2004 [ 22 ] in the analysis of the SCaFoS dataset.

Phylogeny of Nematoda based on the IQ-TREE maximum likelihood analysis of the SCaFoS dataset. IQ-TREE / RAxML bootstrap support values < 100% are shown. Newly generated transcriptomes are marked with an asterisk

With respect to higher-level ecdysozoan (Fig.  3 ) relationships, both analyses recovered Scalidophora (represented by Priapulida + Kinorhyncha) monophyletic and sister to the rest of Ecdysozoa with strong support. IQ-TREE analysis of the PhyloTreePruner dataset recovered Onychophora sister to Arthropoda with strong support (bs = 98%) while the RAxML analysis had only moderate support for this placement (bs = 78%). However, analyses of the SCaFoS dataset recovered Onychophora sister to all non-scalidophoran ecdysozoans with similar levels of support (bs = 100%/82%). IQ-TREE analyses recovered Tardigrada sister to Nematoda with moderate to strong support (bs = 84–97%) whereas RAxML analyses recovered Tardigrada + Nematomorpha sister to Nematoda. This was strongly supported in the analysis of the SCaFoS dataset (bs = 100%) but weakly supported in the analysis of the PhyloTreePruner dataset (bs = 66%).

Phylogeny of outgroup taxa based on the IQ-TREE maximum likelihood analysis of the PhyloTreePruner ( a ) and SCaFoS ( b ) datasets. IQ-TREE / RAxML bootstrap support values < 100% are shown. Newly generated transcriptomes are marked with an asterisk

Deep nematode phylogeny

Early evolution and diversification of nematodes has been a matter of much controversy (reviewed by [ 4 , 15 , 21 , 22 , 54 ]). Molecular phylogenetic studies have generally supported the existence of three major lineages and the monophyly of Chromadoria, but resolution of the deepest splits within Nematoda - relationships among Enoplia, Dorylaimia, and Chromadoria - has been recalcitrant. As in prior analyses based on 18S rDNA [ 7 , 36 , 53 ], analysis of our PhyloTreePruner-based dataset lacked support for relationships among these deepest branches in Nematoda. Enoplia received moderate support (bs = 88), while monophyly of Enoplida could not be established. Insufficient taxon sampling and limited matrix occupancy for Enoplia is, in our opinion, the prime issue to be considered and addressed in efforts to resolve relationships among these deep branches.

Our initial dataset assembly strategy employed PhyloTreePruner [ 49 ], which helps exclude paralogous sequences and contamination missed by the initial orthology inference approach. PhyloTreePruner examines single-gene trees and, if there are two or more sequences from a taxon that do not form a clade, the tree is pruned to the largest subclade in which all taxa are represented by just one sequence. Only the subset of sequences corresponding to that subtree is retained for concatenation and species tree reconstruction. Unfortunately, the PhyloTreePruner algorithm can result in the unnecessary exclusion of large numbers of sequences when even a single taxon has two or more sequences that do not form a clade in single-gene trees (Thálen and Kocot, unpublished data). Aside from paralogy, putative single-gene trees with two or more sequences from the same taxon that do not form a clade may also be caused by the presence of very short and/or mis-aligned contigs, low-quality contigs, or incorrect single gene trees. This problem is exacerbated as the number of sampled taxa increases (Thálen and Kocot, unpublished data).

Use of PhyloTreePruner with its strict orthology inference approach on this rather species-rich dataset resulted in exclusion of large subtrees worth of sequences for many of the orthogroups identified by HaMStR and a final concatenated dataset with just 15.33% matrix completeness. Because the HaMStR “model organisms” core ortholog set used in this study is known to consist of genes that are single copy across diverse metazoan phyla [ 48 ], paralogy is unlikely to be problematic with this dataset (although taxon-specific gene duplications are possible). Thus, we re-ran our pipeline using SCaFoS [ 50 ] to select sequences for concatenation. SCaFoS excludes highly divergent sequences (i.e., it is still able to exclude, non-nematode contamination) and selects the best sequence for each taxon based on average p-distance. As noted above, this resulted in a larger and much more complete dataset (64.99% matrix occupancy).

Despite substantial differences in matrix completeness, analysis of the SCaFoS-based matrix resulted in a very similar topology to that of the PhyloTreePruner-based matrix. Of significance, analysis of this more complete data matrix resulted in strong support for relationships among the major lineages of Nematoda, placing a monophyletic Enoplia sister to all other nematodes with maximal support, and supporting the monophyly of Enoplida. Our SCaFoS-based phylogeny supports the “traditional” view of early nematode evolution with Enoplia sister to the rest of Nematoda, a topology used as a basis for the long-standing yet poorly explored hypothesis that the phylum arose in the marine environment [ 22 , 24 , 26 , 35 ]. The alternative hypothesis of the primarily terrestrial Dorylaimia as the sister to the rest of Nematoda [ 22 ], receives no support from either of our analyses.

Placement of Enoplia as sister to the rest of Nematoda, however, does not deny the possibility of a terrestrial origin of Nematoda [ 22 ] as early-branching clades are equally represented by marine, freshwater and terrestrial taxa (Fig.  4 ). Enoplia splits into predominantly marine Enoplida and predominantly freshwater/terrestrial Triplonchida, while its sister clade (unnamed, containing the rest of Nematoda) consists of primarily marine Chromadoria and primarily freshwater/terrestrial Dorylaimia. A comprehensive hypothesis of nematode origin and early evolution must build on a greatly expanded phylogenomic dataset with better sampling of Enoplia and Dorylaimia and closely related phyla (Nematomorpha, Tardigrada, Priapulida, Kinorhyncha and Loricifera, Onychophora). This would better enable ancestral character state reconstruction analysis for Nematoda and Ecdysozoa as a whole.

Simplified nematode phylogeny based on Fig. 2 indicating marine versus freshwater/terrestrial distribution for each order, considering the distribution of the majority of species. Notes: * includes equal number of marine, freshwater and terrestrial taxa, with molecular phylogenies suggesting terrestrial clades to be earlier (deeper); ** based on distribution of hosts, marine taxa may be of secondary origin; *** based on distribution of hosts; **** based on distribution of hosts and free-living stages

Relationships within major nematode clades

In terms of relationships within major nematode clades, our results are largely consistent with earlier studies based on the 18S rDNA gene [ 27 , 30 , 36 , 53 ] and previous phylogenomic studies [ 40 , 42 ]. One exception is the topology within Dorylaimia, which is somewhat different: 18S rDNA-based trees place Dorylaimida as the earliest branching clade [ 36 , 53 ], although relationships among Mononchida, Mermithida, Trichinellida and Dioctophymatida vary. Our results place a clade containing Dorylaimida, Mermithida and Mononchida sister to a clade with Dioctophymatida and Trichinellida. Our recovery of Mermithida as the sister taxon of Mononchida is in agreement with 18S rDNA based phylogenetic studies (e.g. [ 27 ], but in discordance with morphology-based theories, which suggest closer affinities between Mermithida and Dorylaimida [ 55 , 56 ] or Mermithida and Trichinellida (=Trichocephalida) [ 57 ]. Another exception is in the branching pattern of Rhabditida: our analysis places Spirurina as a sister to Tylenchina + Rhabditina (in full agreement with all 18S rDNA-based and most phylogenomic studies), while [ 42 ] recovered Tylenchina as a sister to Rhabditina + Spirurina, albeit with relatively low bootstrap support.

“Minor” problems in nematode phylogeny

Early radiation within the phylum Nematoda is the most challenging problem but not the only one in the systematics of this group of animals. There are a number of “orphaned” nematode taxa for which phylogenetic affinities and thus placement in the classification remain unclear. Such are the phylogenetic relationships of nematode families Teratocephalidae [ 22 ], Chambersiellidae [ 58 ], Brevibuccidae [ 22 ], Myolaimidae [ 59 ], Aegialoalaimidae [ 60 ], Cyartonematidae [ 61 ], Aulolaimidae [ 62 , 63 ], Paramicrolaimidae [ 60 , 64 ], Haliplectidae [ 60 ], Richtersiidae [ 65 ], Rhabdodemaniidae [ 51 , 66 ], Thalassogeneridae [ 67 ], suborder Ceramonematina [ 60 ] and orders Benthimermithida [ 68 , 69 ], Marimermithida [ 70 ] and Rhaptothyreida [ 71 ]. They often possess unusual morphologies [ 59 , 63 , 64 ] or are highly specialized parasites [ 69 , 70 ], and have no clear place in morphology-based classifications.

Acquisition of transcriptome or genome data from the understudied taxa is needed in order to resolve these “minor” phylogenetic issues that could not be clarified in phylogenetic studies based on rDNA loci or morphology, which have provided contradictory results depending on the data or methodology used. Besides finally achieving stable classification, many of these taxa are important for understanding of morphological character evolution, transitions between marine and terrestrial lifestyles, and evolution of symbiosis in the marine environment.

Phylogeny of Ecdysozoa

Although taxon sampling of the present study focused on Nematoda, we aimed to broadly sample relevant outgroups using only high-quality, publicly available data plus new transcriptomes from a nematomorph and a kinorhynch. Relationships among ecdysozoan phyla have varied somewhat dramatically among studies (reviewed by [ 72 ]), prompting numerous conflicting phylogenetic hypotheses. Our results find no support for some traditionally hypothesized groups including Nematoida (Nematoda + Nematomorpha), Panarthropoda (Arthropoda, Onychophora, and Tardigrada), or Cycloneuralia (Scalidophora + Nematoida). Interestingly, we recover Tardigrada as the sister taxon of Nematoda. A close relationship of Tardigrada to Nematoda has been recovered in other recent phylogenomic studies [ 73 , 74 , 75 , 76 , 77 ], but data from representatives of Nematomorpha have been limited. Interestingly, the PhyloTreePruner-based analysis recovers the traditionally hypothesized placement of Onychophora as the sister taxon of Arthropoda with strong support (bs = 98) but in the SCaFoS-based analysis, it is recovered as the sister taxon of a clade of all other non-scalidophoran ecdysozoans with maximal support. The limited taxon sampling for key ecdysozoan clades (e.g., just one onychohoran, one nematomorph, no heterotardigrades, no loriciferans, etc.) further demonstrates the need for high-quality genomic and transcriptomic resources from this part of the animal tree.

Expand sampling of free-living nematodes to learn more about parasites

The origin and evolution of animal parasitic nematodes from their free-living ancestors has been an active area of research for 80 years [ 78 , 79 , 80 , 81 , 82 , 83 ]. Two simplified scenarios describe evolutionary pre-adaptations and morpho-physiological changes leading towards parasitism via commensalism in aquatic environments [ 84 , 85 ] and via a saprobiontic lifestyle in terrestrial environments [ 80 , 86 , 87 ]. We are just beginning to understand the genomic changes involved in these processes [ 42 , 88 ]. Furthermore, many other important questions about parasite biology remain unanswered, such as how parasites locate and invade hosts, suppress host immune response, acquire nutrients, etc. [ 40 ].

Comprehensive understanding of morphological, ecological, behavioral and genomic adaptations involved in the evolution of a parasitic lifestyle can not be achieved without thorough comparison between parasites and their close, free-living relatives [ 19 , 40 , 89 ]. One of the complications, however, is that animal parasitic nematodes evolved independently at least 18 times [ 90 ], if not more [ 40 , 86 ], and one cannot expect the same underlying mechanism to be behind these numerous independent events. Moreover, the majority of animal parasitic clades have no identified, closely related free-living taxon suitable for comparative analysis [ 19 ]. These include all parasites from the subclass Dorylaimia and the most diverse and economically important Spirurina. Even the closest relative of such a well-researched taxon as the entomopathogenic genus Steinernema remains unclear [ 58 , 91 ]. Thus, further expanding sampling of free-living nematodes in phylogenomic studies will be an integral part of any future research aiming to understand the evolution of parasitism – it will help elucidate sister-group relationships of those parasitic taxa for which the closest free-living relatives are yet unidentified and provide much needed comparative data for identification of parasitism-related genetic modifications.

With 97 published and nine new nematode genomes and transcriptomes, our phylogenetic analyses, which are by far the most comprehensive to date, cover less than 0.5% of the approximately 23,000 valid nematode species [ 92 ]. For comparison, the latest phylum-wide 18S rDNA-based phylogeny [ 53 ] included 1215 sequences or just about 5% of the known diversity. Of the 108 nematode species included in our analyses, 80 belong to Rhabditida – a clade with over 13,400 known species including most economically and medically important parasites as well as the model species Caenorhabditis elegans and satellite model Pristionchus pacificus . Of the Rhabditida species included in our analyses, 50 are parasites of animals, 12 are plant parasitic, and the remaining 18 are thought to be free-living inhabitants of soil or saprophytic communities (although some are phoretically associated with invertebrates). The next largest set of species, 11 in number, represent exclusively the parasitic order Trichinellida (with about 400 known species). The remainder of the phylum, consisting of 17 orders and including free-living (in particular almost all known marine species), plant- and animal parasitic nematodes (with about 9200 species in total), is unevenly represented in our analysis: nine orders are represented by 17 species, while eight orders are not included at all. Out of 108 species included in this phylogenetic analysis, 63 are animal parasitic and 14 are associated with plants, while only 31 are free-living, of which 22 are fresh water and soil inhabitants and only nine are marine. Thus, vast habitat diversity, and the morphological and molecular adaptations that allow nematodes to live in those environments, remains unrepresented in transcriptome-based phylogenies.

Recommended sampling strategies

Three possible sampling strategies to increase and diversify nematode genomic and transcriptomic datasets can be suggested, depending on the research goals. Those researchers who are interested solely in the origin and early evolution of animal parasitism can find interesting models among free-living Enoplida [ 93 ], Chromadorida [ 94 ], Monhysterida [ 95 , 96 ] and Plectida [ 97 , 98 , 99 ] – species with parasitic lifestyles but with morphology retaining many features of their close, free-living relatives. Phylogenetic analysis and subsequent ancestral character state reconstruction would elucidate features of free-living ancestors of parasites and generate new hypotheses regarding the evolution of parasitism. Secondly, studies aimed at improving general nematode phylogeny and classification must focus on the species described above in the “Minor” problems in nematode phylogeny and taxa to which they were once believed to be related to. Finally, large taxonomic categories currently represented by single or few genomes/transcriptomes (Triplonchida, Mononchida, Dorylaimida, etc) also deserve attention, and further sampling of those taxa would elucidate relationships in those clades and likely spur research into yet more unanswered questions.

This study represents the largest phylogenomic analysis of nematodes to date, and furthers our understanding of nematode relationships. We have also, however, revealed how poorly sampled the current dataset is relative to the tremendous diversity of nematodes on Earth. Sequencing and re-sequencing of more species and broad scale comparative studies can also reveal and correct misidentified or mislabelled datasets (the case of Heterorhabditis indica ). Transcriptome sequencing of nematodes is still strongly biased toward parasitic and “model” taxa, particularly those in the Rhabditida, neglecting the free-living clades that hold the key to the origins of the phylum. Our understanding of nematode early evolution and various pathways towards parasitism will be improved only by broader sampling and sequencing of free-living taxa.

Nematodes and the kinorhynch were collected and isolated following standard protocols for sampling meiofauna [ 100 ]. Immediately after isolation, live specimens of Anaplectus granulosus, Euchromadora sp., Symplocostoma sp., and Tobrilus sp. were frozen in 100 μL of nuclease-free water at − 70 °C. Bathylaimus sp., Gordius sp., Odontophora sp., Oncholaimidae sp., Pontonema sp., Pycnophyes sp., and Thoracostomopsidae sp. were preserved in RNA later and stored at − 20 °C.

Total RNA was extracted from all samples but Gordius sp. using the Ambion RNAqueous-Micro Kit. For Anaplectus granulosus, Euchromadora sp. and Tobrilus sp., 1000 μL of lysis solution was added directly to the original sample (nematodes in 100 μl of nuclease-free water), while individual specimens of the remaining nematodes and the kinorhynch were manually transferred from RNAlater or nuclease-free water to lysis solution. Subsequent steps of RNA extraction and DNAse treatment followed the manufacturer’s protocol. RNA was extracted from the nematomorph Gordius sp. using the Omega Bio-Tek EZNA Mollusc RNA kit using a rotor-stator homogenizer for homogenization and on-column DNAse treatment.

For Anaplectus granulosus , Bathylaimus sp., Euchromadora sp., Odontophora sp., Pontonema sp., Symplocostoma sp. and Tobrilus sp., library preparation and cDNA synthesis was performed using the Clontech SMARTer PCR cDNA Synthesis Kit following manufacturer’s instructions. Resulting double-stranded cDNA was purified using the QIAquick PCR Purification Kit. Concentration of double-stranded cDNA was measured using Qubit dsDNA HS Assay Kit and Qubit 3.0 Fluorometer. Final library preparation and transcriptome sequencing were performed at the Swedish National Genomics Infrastructure in Stockholm, Sweden using the Illumina TruSeq PCR-free protocol and an Illumina HiSeq 2500 in high-output mode with V4 2 X 125 bp paired-end reads.

For Oncholaimidae sp. and Thoracostomopsidae sp., total RNA (not quantified; < 1 ng) was sent to Macrogen Inc. (Seoul, South Korea) for cDNA library preparation with the SMARTer low input RNA kit and sequencing using on the Illumina HiSeq 2500 using HiSeq SBS V4 with 2 X 100 bp paired-end reads. For Gordius sp., total RNA (1 μg) was sent to Macrogen for Illumina TruSeq RNA library preparation and sequencing using the Illumina HiSeq 2500 using HiSeq SBS V4 with 2 X 100 bp paired-end reads.

Dataset assembly and analysis followed the approach of Kocot et al. [ 47 ]. Publicly available genomic data [ 101 , 102 ] were downloaded as predicted proteins if available (Additional file 2 : Table S1). Transcriptome dataset of Plectus sambesii was provided by Dr. Philipp Schiffer (CLOE, University College London, UK) and Dr. Christopher Kraus (Zoological Institute, Universität zu Köln, Germany), while transcriptome of Pontonema vulgare was provided by Dr. Andreas Hejnol (Sars International Centre for Marine Molecular Biology, University of Bergen, Norway). Otherwise, predicted transcripts from genomes or assembled transcriptomes were downloaded when possible. After demultiplexing, raw reads for Anaplectus granulosus , Bathylaimus sp., Euchromadora sp., Odontophora sp., Pontonema sp., Symplocostoma sp. and Tobrilus sp. were filtered using AfterQC [ 103 ] and assembled with Trinity [ 104 ] installations available on public Galaxy [ 105 ] servers at usegalaxy.org (Center for Comparative Genomics and Bioinformatics at Penn State, the Department of Biology and at Johns Hopkins University and the Computational Biology Program at Oregon Health & Science University) or galaxy.ncgas-trinity.indiana.edu (National Center for Genome Analysis Support, Pervasive Technology Institute at Indiana University). Pycnophyes sp., Gordius sp., Oncholaimidae sp. and Thoracostomopsidae sp. as well as publicly available transcriptomes available only as raw reads were quality filtered, adapter-trimmed, and assembled using Trinity 2.2.0 with the --trimmomatic and --normalize_reads flags [ 104 ] on the University of Alabama UAHPC cluster. Transcripts were translated with TransDecoder 2.0.0 or 2.0.1 [ 106 ] using the UniProt SwissProt database (accessed on September 20th, 2016; The Uniprot Consortium 2014) and PFAM (Pfam-A.hmm) version 27 [ 107 ].

For orthology inference, HaMStR 13 [ 48 ] was used with the “model organisms” core-ortholog set. Translated transcripts for all taxa except Caenorhabditis elegans were searched against the 1031 profile hidden Markov models (pHMMs) using the “-central” flag and otherwise with the default options. Sequences matching a pHMM were compared to the proteome of Caenorhabditis elegans using BLASTP with the default search settings of HaMStR. If the Caenorhabditis elegans amino acid sequence contributing to the pHMM was the best BLASTP hit in each of these back-BLASTs, the sequence was then assigned to that putative orthology group (simply referred to as “gene” henceforth). Redundant sequences that were identical (including partial sequences that were identical at least where they overlapped) were then removed with UniqHaplo ( http://raven.wrrb.uaf.edu/~ntakebay/teaching/programming/perl-scripts/uniqHaplo.pl ), leaving only unique sequences for each taxon. Each gene was then aligned with MAFFT 7.215 using the automatic alignment strategy with a “maxiterate” value of 1000 [ 108 ]. Alignments were then trimmed with BMGE (−g 0.5) to remove ambiguously aligned regions and any alignments shorter than 50 bp were deleted. Sequences that did not overlap with all other sequences in the alignment by at least 20 amino acids were deleted, starting with the shortest sequences not meeting this criterion. This step was necessary for downstream single-gene tree reconstruction. Finally, genes sampled for fewer than 10 taxa after these steps were discarded.

In some cases, a taxon was represented in an alignment by two or more sequences (splice variants, lineage-specific gene duplications [=inparalogs], undetected paralogs, or exogenous contamination). To screen for evidence of paralogy or contamination and select just one sequence for each taxon, an approximately maximum likelihood tree was inferred for each remaining alignment using FastTree 2 [ 109 ] using the -slow and -gamma options. PhyloTreePruner [ 49 ] was then employed to use a tree-based approach to screen each single-gene alignment for evidence of paralogy or contamination. First, nodes with support values below 0.95 were collapsed into polytomies. Next, the maximally inclusive subtree was selected where each taxon was represented by no more than one sequence or, in cases where more than one sequence was present for any taxon, all sequences from that taxon formed a clade or were part of the same polytomy. Putative paralogs and contaminants (sequences falling outside of this maximally inclusive subtree) were then deleted from the input alignment. In cases where multiple sequences from the same taxon formed a clade or were part of the same polytomy, all sequences except the longest were deleted. Concatenation of remaining sequences to assemble the data matrix henceforth referred to as the “original full dataset” was performed using FASconCAT-G [ 110 ].

Because PhyloTreePruner can result in the unnecessary exclusion of large numbers of sequences when even a single taxon has unstable or contaminant sequences, we also ran our pipeline using SCaFoS [ 50 ] instead of PhyloTreePruner. The default settings were used to exclude highly divergent sequences and select the best sequence for each taxon based on average p-distance to all other sequences in the alignment.

Maximum likelihood (ML) analyses were conducted in RAxML 8.2.8 [ 111 ] and IQ-TREE [ 112 ]. Because of the very large number of taxa in our matrices, for the RAxML analyses, data matrices were partitioned by gene but the PROTGAMMALG model was specified for all partitions rather than empirically inferring the best-fitting model for each partition. A preliminary run of PartitionFinder 2 [ 113 ] found that the LG model was the best fit for the vast majority of partitions. The tree with the best likelihood score after 10 random addition sequence replicates was retained and nodal support was assessed with rapid bootstrapping with the number of replicates determined by the autoMRE criterion. IQ-TREE analyses were performed using IQ-TREE 1.5.5 with the site heterogeneous PMSF model [ 63 ] (−m LG + C20 + G + F) with the RAxML bipartitions tree provided as the required guide tree (−ft). Nodal support was assessed with 1000 rapid bootstraps (−bb 1000).

Several taxonomy assignment approaches were used to identify the transcriptome of Heterorhabditis indica . At first, transcriptome database for Heterorhabditis indica available at http://insilico.iari.res.in/hindica/ was mined for possible ribosomal DNA sequences using built-in BLAST search and 18S rDNA sequence of Plectus aquatilis (chosen to be equally distantly related from both Heterorhabditis and Heterodera ) as a target. Four recovered transcripts were then compared with the publicly available sequences from the nucleotide collection of NCBI GenBank using blastn suite (alignment-based taxonomy assignment approach, see review in [ 114 ]). One of the recovered transcripts (labelled as Locus_123_Transcript_1/1) showed high similarity (> 99% identity, E-value = 0) to several 18S sequences from different species of the genera Heterodera and Globodera , with Heterodera glycines (GenBank acc. Number AY043247) having the highest identity score, albeit with partial overlap. The other two transcripts (labelled as Locus_90_Transcript_1/2 and Locus_90_Transcript_2/2 respectively) also showed high similarity (99% identity, E-value = 0) to several sequences from different species of the genera Heterodera and Globodera , partially overlapping various reference sequences that may include partial 18S, ITS1, 5.8S, ITS2 and partial 28S, with Heterodera cajani (GenBank acc. Number AY274389) having the highest identity score. Similar results were obtained by mining the transcriptome assembly downloaded directly from GenBank.

The longest section of 18S rDNA sequence mined from the Heterorhabditis indica transcriptome database (588 base long partial 5′ section from the Locus_123_Transcript_1/1) was then used in tree-based taxonomy assignment approach (see review in [ 115 ]) to double-check the results of alignment-based taxonomy assignment. This section was added to a selection of 18S rDNA sequences downloaded from SILVA database [ 116 ] and representing all major clades of Rhabditida, including all available near-full length sequences for identified species from the genera Heterorhabditis, Heterodera and Globodera. The alignment was created using MUSCLE at https://www.ebi.ac.uk/Tools/msa/muscle/ under default settings and trimmed to a size of a fragment from the Heterorhabditis indica transcriptome. A phylogenetic tree was inferred using RAxML-HPC2 under default settings with 1000 bootstrap replicates.

Availability of data and materials

The datasets generated and analysed during the current study including transcriptome assemblies are available via FigShare: https://figshare.com/s/4c8e501714dbd5be1be8

Abbreviations

Bootstrap support

Maximum likelihood

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Acknowledgements

OH acknowledges support from the National Genomics Infrastructure funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. OH also acknowledges the use of public servers usegalaxy.org (Center for Comparative Genomics and Bioinformatics at Penn State, the Department of Biology and at Johns Hopkins University and the Computational Biology Program at Oregon Health & Science University) and galaxy.ncgas-trinity.indiana.edu (National Center for Genome Analysis Support, Pervasive Technology Institute at Indiana University) for analysis of some of the data. Sampling in the Skagerrak was conducted by OH using vessels (“Skagerak” and “Oscar von Sydow”) and facilities of the Sven Lovén Centre for Marine Sciences in Kristineberg. Further thanks are to Dr. Sarah Atherton (Swedish Museum of Natural History) who collected samples containing Euchromadora sp. and Symplocostoma sp. KMK thanks Dr. Christoph Held, the Alfred Wegener Institute, and the scientists and crew of the R/V Polarstern PS 96 cruise, which provided samples used in this work. KMK also thanks Deb Crocker and Robert Griffin for assistance with the University of Alabama High-Performance Computing cluster. Dr. Andreas Hejnol (SARS Centre, University of Bergen), Dr. Philipp Schiffer (CLOE, University College London), and Dr. Christopher Kraus (Zoological Institute, Universität zu Köln) kindly provided unpublished transcriptome data. The authors gratefully acknowledge use of the resources of the Alabama Water Institute at The University of Alabama.

AS received funding from the Virginia Military Institute to support field work, identification of specimens, and manuscript preparation. Nematode sampling and transcriptome sequencing was supported primarily by two grants to OH from the Swedish Taxonomy Initiative, Artdatabanken: “Systematics of Swedish free-living nematodes of the orders Desmodorida and Araeolaimida” (Dha 2013–140) and “Systematics of poorly known marine nematodes of the class Chromadorea from Sweden” (SLU.dha.2017.4.3–102), and by a grant from Riksmusei Vänner: “Transcriptome sequencing of marine nematodes.” KMK received funding from The University of Alabama to support field work, lab work, transcriptome sequencing, data analysis, and manuscript preparation.

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Oleksandr Holovachov

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KMK, OH, and AS collected specimens, OH and AS identified specimens, KMK and OH performed RNA extraction and library preparation, and KMK analyzed the data. All authors contributed to the writing of the manuscript and approved the final version prior to submission.

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All specimens used in this study were collected from wild populations. Field work was conducted in accordance with local regulations. Antarctic nematodes and the kinorhynch were collected during R/V Polarstern cruise PS96 with permission from the Alfred Wegener Institute, Germany. No permits were required to collect any of the other taxa sequenced in this study.

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Additional file 1:.

Figure S1. Phylogenetic position of 18S rDNA fragment (contig Locus_123_Transcript_1/1) extracted from Heterorhabditis indica transcriptome dataset ( http://insilico.iari.res.in/hindica/ ). (PDF 46 kb)

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Table S1. Names, classification (family and order), accession number (BioProject) or download link, and source/citation for publicly available genomes and transcriptomes of nematodes used in phylogenetic analysis. Classification is based on [ 1 ] with modifications [ 2 , 3 ]. Table S2. Names and origin data or reference for publicly available genomes, transcriptomes or proteomes of non-nematode (outgroup) taxa. (DOCX 22 kb)

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Smythe, A.B., Holovachov, O. & Kocot, K.M. Improved phylogenomic sampling of free-living nematodes enhances resolution of higher-level nematode phylogeny. BMC Evol Biol 19 , 121 (2019). https://doi.org/10.1186/s12862-019-1444-x

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DOI : https://doi.org/10.1186/s12862-019-1444-x

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Screening the nematicidal potential of indigenous medicinal plant extracts against Meloidogyne incognita under lab. and greenhouse conditions

• Hosny Kesba 1 ,
• Abdullah Abdel-Rahman 1 ,
• Samy Sayed   ORCID: orcid.org/0000-0002-7002-568X 2 &
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Egyptian Journal of Biological Pest Control volume  31 , Article number:  81 ( 2021 ) Cite this article

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The root-knot nematode, Meloidogyne incognita , causes a high damage and yield decrease for many economic plants. The need for non-systemic effective new approaches and environmentally friendly methods for controlling the nematodes has directed research to some new and safe agrochemicals found in medicinal plants as new viable management options.

In laboratory experiments, solidago and periwinkle aqueous and ethanolic extracts achieved high J2 mortality (%) concerning different dilutions; however, aqueous extracts were more effective for mortality than ethanolic extracts. Also, there was a direct relationship between the nematicidal activity of these extracts with both concentration and time of application. Inhibition of egg hatching by Periwinkle extracts was higher than that of solidago. Moreover, the nematicidal activity of tested extracts against J2 decreased significantly with prolonged storage time at + 5 °C, while did not with stored frozen at – 5 °C for 12 months. Periwinkle and solidago extracts killed the non-target organisms, i.e., rotifers and free-living nematodes. Seventy-five and 90% of total phytochemicals recovered from periwinkle and solidago, respectively were nematostatic or nematicidal to nematode viability, egg hatch in vitro, and development and reproduction in vivo despite the method of application (foliar and soil drench). The antagonistic effects of solidago were more pronounced in soil drench than periwinkle concerning their concentrations and methods of application.

Solidago and periwinkle plant extracts showed important sources of effective control phytochemicals against M. incognita .

Botanicals can be used in in vitro and in vivo by different ways as one of the nonchemical approach strategies to manage and reduce plant-parasitic nematodes, especially in sustainable agriculture (Bridge 1996 ) by using their parts directly, their extracts, and compounds that possessing nematicidal activities, oilseed cakes, mature crop residues as organic amendments (Manju and Meena 2015 ). Some of the botanicals are already being exploited commercially in pest management and a rising trend towards organic farming (Zaidat et al. 2020 ).

In vitro, a lot of plant extracts showed high ovicidal and nematicidal effects on egg hatching and J2 survival of the root-knot nematode (RKN), M. incognita . Extracts from Nicotiana tabacum , Syzygium aromaticum , Piper betle , and Acorus calamus were found more effective in killing M. incognita , with an EC 50 which was 5–10 times lower than the EC 50 of the synthetic pesticides, chlorpyrifos, carbosulfan, and deltamethrin (Taniwiryono et al. 2009 ). The nematicidal effect of plant extracts could be higher than synthetic nematicides.

In vivo, (under greenhouse or field conditions) application of plant extracts reduced infection of RKN nematode and caused crop yield increase. These extracts were more effective than the nematicides used or in the same order or slightly less.

Dozens of phytochemical compounds that may be more active and eco-friendly, especially those came from medicinal and aromatic plants, e.g., serpentine, saponins, phenols, alkaloids, tannins, flavonoids, steroids, and cysteine proteinases have been reported for their antihelmintic effect against human, animal, and plant parasites (Rocha et al. 2017 ). The nematicidal effects of dried parts and boiled extract of Bidenspilosa were bioactive when re-evaluated on phytoparasitic nematodes after storing up to 12–18 months (Taba et al. 2012 ).

This study focused on the nematicidal potential of some medicinal plant extracts in the management of root-knot nematode in vitro and in vivo and their preservation on nematicidal activity, non-target organisms, and chemical composition.

Nematode culture

Pure stock culture of the RKN, M. incognita originally obtained from galled eggplant roots was established. Single egg-mass from previously identified females (Taylor et al. 1955 ) was used to inoculate healthy eggplants grown in (20 cm diameter) earthen pots filled with loamy sand soil. Three months after inoculation, plants were examined for nematode infection and reproduction. The culture was propagated and maintained on eggplant.

Plant extracts preparation

The effect of aqueous and ethanolic extracts of 13 medicinal plants was evaluated directly or after storage periods for lethal concentrations and toxicity index on survival and hatchability of M. incognita and non-target organisms’ management under laboratory conditions (in vitro). Therefore, foliar spray and soil drench applications of solidago and periwinkle, which achieved the highest mortality percentages than the other tested plant species were carried out in the greenhouse (in vivo) on infected sunflower plants.

Aqueous extracts

Twenty-five grams of air-dried leaves of 13 medicinal plants listed in Table  1 were homogenized by grinding for 1 min using a blender to coarse particles (formation of extremely soft particles like a powder that may hamper better extraction was avoided by the solvent as described by Pandey and Tripathi 2014 ). Then, 1 L of tap water was added. The mix was transferred to a 2-L beaker and was shaken vigorously. Decoction process was done to extract water-soluble and heat-stable constituents, in which the mix was boiled for 10 min, cooled, and filtered using filter paper. This mix was kept frozen as a stock solution (1×) until use. The stock solution was diluted by adding tap water to prepare the diluted extracts. Seven extracts dilutions (1:2×, 1:4×, 1:8×, 1:16×, 1:30×) were prepared by adding sufficient tap water to the stock solution till reaching the required concentrations. The 5 dilutions were equivalent, respectively, to the 5 concentrations (12500, 6250, 3120, 1560, 780 mg dry weight/liter (mg D.Wt./L)).

Ethanolic extracts

Five grams of the previously mentioned medicinal plants’ air-dried leaves were homogenized to coarse particles using a blender, then added to 200 ml of ethyl alcohol 96% in a 1-L beaker, shaken for 24 h using a shaker at room temperature, and filtered using a filter paper. A rotary evaporator was used to evaporate the solvent (ethanol) under vacuum to prepare the crude extracts, which then were dissolved in 5 ml ethanol and added to 200 ml tap water + 1 ml tween 80 as a surfactant. The resulting solution was shaken and kept frozen as a stock solution (1×). This stock solution is equivalent to a concentration of 25 mg D.Wt./L. Only 4 dilutions (1/2×, 1/4×, 1/8×, and 1/16×) were prepared by adding tap water to the stock solution till reaching the required dilutions, equivalent to 12500, 6250, 3120, 1560 mg D.Wt./L concentrations, respectively.

In vitro tests

Approximately 800 newly hatched J2 of M. incognita were tested for survival after exposure to the mentioned plant extracts after 48 h. For each treatment, 5 replicates were prepared in test tubes and kept under room temperature conditions. Juveniles in tap water only were served as a check. Mobile and immobile nematode J2s were counted under the microscope. Dead (immobile) J2s gave different strange body shapes such as S, Curly shapes. High protozoan and metazoan activities were noticed after juveniles’ death. Also, there was great degeneration ‘shrinkage’ starting after the stylet base and along the esophagus of the dead juveniles. Reversible effects were not expected. Mortality percent was calculated by the following formula:

Calculating lethal concentrations and toxicity index for M. incognita J2

Data of mortality percentages (%) in vitro were input to LDP line software to calculate probit analyses according to Finney ( 1971 ), which was used to illustrate the relation between stimulus and response in toxicological studies. The toxicity index of each plant extract was determined according to Sun ( 1950 ) using the following formula:

Egg hatching inhibition

According to mortality rates of J2 in the last-mentioned in vitro survival test , only 2 aqueous extracts that caused the highest mortality rates were chosen to test their effect on egg hatching rates, those were solidago and periwinkle extracts. Full egg masses of M. incognita were teased from infected eggplant roots under the stereomicroscope. Four concentrations of the 2 extracts were tested; 500, 1000, 2000, and 4000 mg D.Wt./L. Each replication received 5 full egg masses in a test tube, 5 replicates for each treatment, and incubated under room temperatures. The check was egg masses in tap water only. One week later, replicates were examined under the microscope to count hatched J2s. Inhibition rates were calculated according to the following formula:

Effects on non-target organisms

Concentrations of 1000 and 2000 mg D.Wt./L of the periwinkle and solidago extracts were tested against 2 kinds of metazoans; Rotifers and free-living nematodes in vitro. Four replicates for each treatment were set and mortality percentages were calculated after 48 h. Each replicate contained a mix of approximately 80 free-living nematodes and 100 rotifer individuals. The population of free-living nematodes was obtained from a soil sample rich in organic matter. However, rotifers were obtained from a soil sample kept at room temperature for a month to increase rotifers counts. The mixed population in water was kept as a check.

Storage periods of aqueous extracts

Samples of freshly prepared stock of each of solidago and periwinkle leave extracts were stored either under freezing at – 5 °C for 1 year, cooled for 2 weeks or 2 months at + 5 °C, then they were re-evaluated for their nematicidal effect changes. The experiment procedures and mortality percentages were done as previously mentioned on M. incognita J2 in vitro.

Greenhouse experiments

Based on data obtained from the mortality in vitro tests on M. incognita J2s , solidago and periwinkle aqueous extracts were tested under greenhouse conditions for M. incognita control. Seeds of sunflower (Giza-102) plants were sown in 220 pots, each filled with 2 kg sandy clay soil (1:1, v:v). Two weeks later, pots were divided into two main groups which were treated as follows:

Foliar spray application

The aqueous extracts were applied as a foliar spray once. Foliar spray drift to the soil was avoided by covering it, using a tissue paper. The tested plant aqueous extracts concentrations were 500, 1000, and 2000 mg dry leaves/L (mg D.Wt./L). Each treatment contained 5 replicates. After 2 weeks of germination, plants were inoculated with 2000 M. incognita J2s/plant . The whole experiment was set and horticultural-maintained for 45 days after nematode inoculation. Only one factor was different for each of the 3 sub-groups, which was the time application, as follows: the first subgroup; extracts were foliar sprayed 1 week before nematode inoculation, the second sub-group; extracts were foliar sprayed simultaneously with nematode inoculation, and the third sub-group; extracts were foliar sprayed 1 week after nematode inoculation. Five replicates in each sub-group received the same treatments, except that instead of applying plant extracts, the synthetic pesticide formulation, Vydate® 24% SL (oxamyl) was foliar sprayed as a standard chemical nematicide (3 ml/L). Another 5 replicates were inoculated with nematode only and kept as a check.

Soil drench application

The aqueous extracts were applied as a soil drench once (100 ml/plant). The tested plant aqueous extracts (solidago and periwinkle dry leaves extracts) concentrations were 500, 1000, and 2000 mg dry leaves/L (mg D.Wt./L). This group contained 110 pots, each treatment contained 5 replicates. Inoculum level was 2000 J2 of M. incognita after 2 weeks of germination . One factor was different for each of the 3 sub-groups, which was the time of extracts application as follows: the first subgroup; 100 ml of extracts solution were applied as a soil drench for each pot 1 week before nematode inoculation, the second sub-group; 100 ml of extracts were applied simultaneously with nematode inoculation, and the third sub-group: 100 ml of extracts were applied 1 week after nematode inoculation. Five replicates in each sub-group were received the same treatments except that, instead of applying plant extracts, the synthetic pesticide formulation, Vydate® 24 % SL, was soil drenched as a standard chemical nematicide (0.1 ml/L). Another 5 replicates were inoculated by nematode only and kept as a check.

Nematode assay

Upon harvest, each pot was soaked in a plastic bucket filled with water until the root system could be easily separated. Each root system was weighed and stored in 5% formaldehyde in plastic jars. The soil suspension was quite stirred and then poured through a series of 60 and 325 mesh screens (Hooper et al. 2005 ). The bottom sieve was then poured onto a modified Baermann set and collected after 48 h. Hawksley counting slide was used to calculate the number of J2s in 1 ml of the suspension and then referred to the whole volume. The numbers of galls and egg-masses were counted directly on the root system of each replicate and the mean of each treatment was calculated and later the eggs were extracted (Boneti and Ferraz 1981 ). For calculating eggs per egg mass, 10 full egg masses from each replicate were chosen, gelatin matrix was dissolved using sodium hypochlorite (NaOCl) according to (Hussey and Barker 1973 ), and eggs were counted in 1 ml volume under the microscope. The final population (eggs + soil population) plotted in the formula RF = Pf/Pi, where RF is the reproduction factor, Pf the final population, and Pi the initial population (Oostenbrink 1966 ).

GC/MS/MS analysis

Sample preparation.

Five grams of grinded dried leaves of each plant (periwinkle and solidago) were added to 100 ml deionized water in a 250-ml flask. The decoction process was done as previously mentioned. The extract was filtered, using filter paper, centrifuged at 9000 rpm for 5 min to exclude any impurities, then it was lyophilized (freeze-dried).

Chromatographic analysis

Produced powder from the last step was analyzed using gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry (GC/MS/MS). The analysis was carried out using a GC (Agilent Technologies 7890A) interfaced with a mass selective detector (MSD, Agilent 7000) equipped with a polar Agilent HP-5ms (5%-phenyl methyl polysiloxane) capillary column (30 m × 0.25 mm i. d. and 0.25 μm film thickness). The carrier gas was helium with a linear velocity of 1 ml/min. The injector and detector temperatures were 200 and 250 °C, respectively. The volume injected 1 μl of the sample. The MS operating parameters were as follows: ionization potential 70 eV, interface temperature 250 °C, and acquisition mass range 50–800.

The identification of components was based on a comparison of their mass spectra and retention time with those of the authentic compounds and by computer matching with NIST and WILEY library as well as by comparison of the fragmentation pattern of the mass spectral data with those reported in the literature (Dong et al. 2014 ).

Statistical analysis

Data were statistically analyzed one-way ANOVA according to the SPSS software package version 23. The differences between means were tested using Duncan’s multiple tests at the 5% significance level.

Effect of aqueous extracts on J2 mortality

Dry leaf powder extracts of 13 medicinal plant species were tested in a laboratory assay on M. incognita J2s’ mortality with 5 dilutions of each. In general, after 48 h of exposure, all extracts used had nematicidal action against M. incognita J2 depending on the plant species and rate of the aqueous extract dilution (Table  2 ). The percent of mortality decreased by increasing extracts dilutions, except that of solidago and periwinkle dry leaf extracts. However, the first 2 dilutions 1:2× and 1:4× were highly toxic in all extracts achieving 100% mortality. Leaf extracts of solidago and periwinkle displayed the highest toxicity in (1:16× and 1:30× dilutions) than the rest of medicinal extracts, although, the percentage of mortality decreased down to almost 70% at (1:30× dilution). A considerable decrease in J2 mortality appeared by (1:16× dilution), less than 50% of J2s were killed by Common mint, horsemint, geranium, and dill extracts. The least percentages of mortality at (1:30× dilution) were recorded by extracts of marjoram, chamomile, and geranium, which were almost similar to the acceptable natural death percentage in the check (without treatment).

The relative toxicity index was measured by calculating and comparing each J2’s mortality relative to the maximum percentage of mortality. The accumulative percentages of mortality were calculated as well and were the highest in periwinkle followed by solidago and the least was found in Fros dill. The toxicity index, as well as the accumulative percentage of mortality, followed the same trend where the highest toxicity index was gained by solidago extract, followed by periwinkle and marjoram, respectively. While dill recorded the lowest relative toxicity value.

Concerning the lethal concentration needed to achieve LC 50 , it was found that solidago recorded the lowest lethal concentration that achieved such criteria and it was almost 1/2:1/3 of most extracts concentrations, followed by periwinkle. Geranium and dill needed four times concentrations as much as solidago to attain LC 50 . LC 90 was found to be achieved by a concentration of almost 1 g D.Wt./L in solidago extract. The lowest lethal concentration for LC 90 was raised to 5 times in horsemint, 10 and 11 times in rosemary and thyme extracts, respectively. Solidago and periwinkle extracts recorded almost the same lethal concentration required to achieve LC 90 .

Effect of ethanolic extracts on J2 mortality

The ethanolic extracts of the previously mentioned medicinal plant species were tested on M. incognita J2s mortality at 3 dilutions. After 48 h exposure, the ethanolic dry leaf extracts of all medicinal species were found lethal to M. incognita J2s, regardless of the rate of dilution, although the mortality percentages differed according to plant species extract and dilution (Table  3 ). There was direct proportional relation to a great extent between mortality and dilution. Considerable high mortality rates were noticed at 1:2× dilution in most cases than 1:4× or 1:8× dilutions. Coriander, geranium, solidago, marjoram, basil, and periwinkle extracts achieved higher mortality in 1:4× dilution than that of 1:2× or 1:8× dilutions. The general decrease in J2 mortality was recorded in 1:8× dilution, when compared with the others. The accumulative mortality percentages of geranium, marjoram, and solidago were the highest meanwhile; horsemint, chamomile, and dill were the lowest. However, the toxicity index of solidago extract was in the lead of all extracts.

From the results, it seems that the aqueous extracts of dry leaves of medicinal species were much more efficient in being nematoxic or nematicidal to M. incognita J2s than ethanolic extracts in all dilutions and that was reflected positively on the least lethal concentrations required for LC 50 and LC 90 .

Effect of storage aqueous extracts against J2

Results of the previous in vitro experiment verified that extracts of solidago and periwinkle recorded the ultimate percentages of J2 mortality amongst all the tested aqueous extracts. So, the effect of storage conditions and temperature on extracts stability and activity and in turn, their immobilization activity was studied (Table  4 ). The storage time affected the extract’s capability on mortality. The nematoxic effects of extracts were match able to those of expected fresh after 14 days storage in the fridge at + 5 °C, but when the storage period was prolonged up to 60 days, the stability of the phytochemicals was drastically affected, which in turn minimized mortality in solidago aqueous extract treatments. That minimization was more pronounced at 1000 mg D.Wt./L concentration. The activity of periwinkle extract lower concentration was less effective than the higher one. The activity of extracts differed after 12 months of freezing conditions at – 5 °C. Periwinkle activity was stable achieving 92% mortality at 1000 mg D.Wt./L and heightened up to 100% at the higher concentration. The opposite was found to be the case in solidago extract lower concentration (78% mortality). The higher concentration was highly effective as much as expected fresh or 14 days cooled storage. It seems that J2s mortality retrogrades proportionally with the time of extract storage. The efficacy of treatment was improved by doubling the concentration but not with the time of storage.

Effect of aqueous extracts on egg hatching

Solidago and periwinkle aqueous extracts which were found to be the most efficient on M. incognita J2s mortality were selected to study their effect on hatching of egg masses at 3 concentrations after 7 days of exposure. Significant inhibitory effects on the numbers of hatched eggs when compared with the check were observed (Fig.  1 ). Also, significant differences were found between and within treatments. Periwinkle extract was significantly efficient in reducing the number of hatched eggs at all concentrations. Non-significant differences were found between the lowest and middle concentrations of periwinkle. There was a significant positive correlation between the concentration of each aqueous extract and the rate of inhibition. The highest rate of inhibition was recorded at periwinkle (98.9%), followed by solidago (87.5%) with the highest concentration (1000 mg D.wt./L). The lowest inhibitory effects were found in the case of the lowest concentration of solidago extract (23.4%).

Efficacy of periwinkle and solidago aqueous extracts at three concentrations on egg hatching of M. incognita egg masses. Columns bear different letters that are significantly different at p < 0.05 according to Duncan’s multiple range tests

Effect of periwinkle and solidago aqueous extracts on non-target organisms

A further in vitro experiment was conducted to study the effect of solidago and periwinkle dry leaf aqueous extracts at 2 concentrations (1000, 2000 mg D.Wt./L) on the free-living nematode genera and rotifers, which were found co-inhabiting the soil with the root-knot nematodes. The recessive effects on both microorganisms were observed regardless of the type of plant extract as well as concentration; however, solidago extract was more efficient than periwinkle were obvious in Fig.  2 . Effects on rotifers were more retrograded than free-living nematodes. Mortality of rotifers after 48 h treatment was consistent in periwinkle leaf extract at the two used concentrations, which achieved more than 50% mortality. It is interesting to notice that percentage of mortality in solidago treatments was directly correlated with concentration, 66.5 at the lower and 84.7 at the higher. On the other hand, solidago aqueous extract interpreted almost similar mortality percentages at the lower and higher concentrations on free-living nematodes. Periwinkle extract at 1000 mg D.wt./L recorded mortality values on free-living nematodes 10% less than its higher concentration which was close to solidago extract concentrations.

Efficacy of periwinkle and solidago aqueous extracts on the mortality of free-living nematodes and rotifers after 48 h in vitro . Columns of the same organism bear different letters are significantly different at p < 0.05 according to Duncan’s multiple range tests

Chemical content of solidago and periwinkle dry leaves

Quantitative and qualitative analysis of phytochemicals in dry leaves of periwinkle and solidago was carried out by GC-MS-MS. The leaf extract manifested four main phytochemical classes. Flavonoids, terpenoids, phenolics, and coumarins were highly present with different concentrations. Flavonoids were the most dominant in all the extracts, followed by the terpenoids, phenolics, and coumarins in the solidago leaf extract. Phenolic compounds were the maximum class found in periwinkle leaves (59.2%), followed by flavonoids (19.7%) and coumarins (5.2%). The minimum was piperazines (3.9%), glycosides (2.6%), and phytosterols (2.4%) in solidago leaves, respectively. Meanwhile, the lowest ratios were organic alcohol (4%), fatty acids (4.1%), alkaloids (3%), and sesquiterpenoids (3%) in periwinkle leaves. There were correspondingly prohibiting effects of the main chemical classes and the ratios on nematode activity, egg inhibition and development, and reproductively.

Effect of periwinkle and solidago aqueous extracts on RKN infecting sunflower

Potentials of periwinkle, solidago dry leaves aqueous extracts under 3 intervals (pre, with, and post-inoculation), 2 methods of application (foliar spray, soil drench) with 100 ml of each of the concentrations (500, 1000, 2000 mg D.Wt./L), and the comparable nematicide Vydate® 24% SL on M. incognita in greenhouse experiment was detected and illustrated in Tables  5 and 6 .

Apropos of pre-inoculation, periwinkle, and solidago extracts significantly suppressed the numbers of formed galls, egg masses, and eggs/egg mass. Except for periwinkle extracts, the numbers of eggs exceeded the nematode check and consequently reflected on nematode reproduction, which was more or less the nematode check. Solidago treatment concentrations performed significant impressive reductions in all nematode criteria and exhibited smashup in nematode final population and buildup. Obvious direct proportional effects were noticed among solidago concentration. Very few eggs were laid in the highest sprayed concentration (2000 mg D.Wt./L) achieving the lowest buildup (Table  5 ).

As for inoculation abreast with foliage spray, all treatments concentration imposed significant smashing reductions on all nematode criteria either intra- or interspecific treatments except the foliar sprays of solidago at 500, 1000 mg D.Wt./L at the number of deposited eggs and buildup, where the nematode was able to fold only once with non-significant differences with the check. Periwinkle concentrations were the most invincible where the least buildup rates were performed.

The post-inoculation treatment followed the same trend with the abreast one, all treatments were significantly effective in reducing numbers of galls, egg masses, final population, and the subsequent buildup and egg production when compared with the check. Solidago and periwinkle at 1000, 2000 mg D.Wt./L outmatched Vydate® 24% SL.

Concerning the foliar spray treatment, Vydate® at all intervals, the nematode galls, egg masses, and fecundity were sharply declined. Values of buildup were eliminated with significant differences where the nematode was not able to fold even once in pre- and post-inoculations.

Efficacies of the aqueous extracts of periwinkle and solidago on M. incognita at different intervals and same concentrations as previously mentioned and treated as a soil drench to sunflower plants are given in Table  6 . It contended in general that soil drench applications were more effective in reducing nematode criteria than foliar spray as measured by gall formation, egg masses fecundity, and buildup. Referring to the used concentrations, most if not all extracts concentrations were highly suppressive for nematode parameters. Solidago extracts surpassed to great extent periwinkle extracts at all intervals and concentrations. Controvert effects were noticeable in periwinkle extracts at 500, 2000 mg D.Wt./L at pre- and post-inoculation intervals, where the nematode was able to fold more than once as compared with the untreated inoculated check. The detersive effects of Vydate® were more malignant when treated as soil drench than foliar spray and in particular when treated, with inoculation, no J2s were able to invade the roots.

GC/MS/MS analysis indicated the presence of phytochemicals belonging to the following classes: phenols, flavonoids, triterpenoids, coumarins, alkaloids, and glycosides. groups with different concentrations. Solidago and periwinkle extracts were in the lead of all the 13 tested extracts in achieving the highest percentages of mortality. Ethanol extracts were effective in increasing mortality to M. incognita J2s but not as much as aqueous extracts. This may be due to the polarity of the solvent; the polarity of water is higher than that of ethanol. Consequently, higher antioxidants and phenols were extracted using water. Ng et al. ( 2020 ) reported that polar solvents could extract higher amounts of antioxidants and phenolic compounds, which increase the radical scavenging activity of the extracts. Neeraj et al. ( 2017 ) reported that alcoholic extracts showed a high activity in immobilization of M. incognita J2s and egg hatching inhibition in vitro. That may be because they used high concentrations.

The nature of medicinal plant structure and their derivatives have been discussed extensively but the mode of action of most nematicidal phytochemicals is still ambiguous. Periwinkle achieved more inhibitory effects on egg hatchability than solidago extract. That inhibition was proportionally correlated with concentration increase. It is well known that periwinkle extracts are rich in alkaloids. Extracts that contain alkaloids were found to have ovicidal property against Meloidogyne eggs (Adegbite 2003 ). Also, Alkaloids may act on the central nervous system and cause paralysis (Roy et al. 2010 ), which gave nematicidal effects. Important notice should be considered when comparing the efficacy of ethanolic and water extracts; solidago extracts with both solvents were the most toxic on J2. However, Periwinkle extract nematicidal effects were pronounced only in the aqueous extract and reduced greatly in the ethanolic one. Herein, the nematicidal and ovicidal effects of periwinkle extract are basically due to the water-soluble fractions.

Solidago aqueous extract had a higher percentage of compounds known with their nematicidal activity than periwinkle extract; such as terpenoids (Ohri and Pannu 2009 ), glycosides (Pronar 1983 ), and piperazines. Actually, for a long time, piperazine derivatives are used as antihelmintic drugs for humans (Shafei et al. 1955 ).

There is a real need for fractionation of the constituents of the extract to test each compound individually. However, one can generalize that the bioactivity against nematodes of each extract follows a multi-site mode of action. This is simply because that there is a large number of compounds in each extract, and these compounds have different functional groups with different modes of action.

Flavonoids, low molecular weight secondary metabolites have diverse functions including defense and auxin transport inhibition, and are implicated in resistance to both sedentary and migratory nematodes (Baldridge et al. 1998 ). Also, hypersensitive response and accumulation of the phytoalexin glyceollin, a product of isoflavonoids inhibit oxidation, respiration of M. incognita in vitro and accumulate adjacent to the head region of soybean cyst nematode in resistant root tissues (Kaplan and Keen 1980 ).

Phenolic compounds interfere with the energy generation mechanism by uncoupling the oxidative phosphorylation and interfere with glycoprotein of the cell surface of the parasite and cause death (John et al. 2009 ). Glycosides nematicidal effects are due to their function as a cholinesterase inhibitor that prohibits the normal buildup of nematode (Pronar 1983 ).

It has been found that prolonging the time of storage of extracts affects their stability and minimizes activity on nematode mortality, which was also influenced by the extract type. Aqueous extracts of periwinkle and solidago could be stored for 12 months without significant loss of nematicidal activity against M. incognita . The boiled extracts of Bidenspilosa could be stored for 12–18 months without loss of nematoxic activity (Taba et al. 2012 ).

Periwinkle and solidago extracts showed toxic effects on the free-living nematode and rotifers in vitro. Logically it may be lower than that of the synthetic nematicides but plant extracts are still considered xenobiotics and could alter the normal activity of the microfauna in the soil. Storage experiment revealed that bioactive compounds in these extracts were stable only at freezing temperatures, so biodegradation of these extracts in the soil is expected in a short period.

The greenhouse results emphasized the toxicity action of periwinkle and solidago extracts either foliar spray or soil drench treatment under different intervals on M. incognita parameters. The negative effects of solidago were more pronounced than periwinkle in most concentrations, method as well as the time of application. The nematicidal phytochemicals represented 90% of the total phytochemicals recovered from solidago meanwhile, that percentage was 75% for periwinkle. The nematode reduction outcomes may be due to the toxic contact action to the nematode juveniles and to the small molecular weight phytochemicals, which absorbed by roots easily that raise the plant resistance against nematodes or may have systemic nematicidal action as proven by pre-, with, and post-inoculation applications. That systemic nematicidal action may be attributable to the high ratios of phytochemical components (Archana and Parasad 2014 ) on other plants with different concentrations. Flavonoids could help in plant resistance against nematodes by affecting chemotaxis towards roots and interfere with functions in nematode reproduction (Chin et al. 2018 ). They added that the mechanism of these effects is still unknown. This can interpret the decline in reproduction factor and number of galls due to the application of both extracts. The medicinal plant extractions treated as soil drench showed significant reducing effects and nematicidal activity to M. incognita criteria (El-Nagdi and Youssef 2013 ). The described effects on M. incognita were achieved by using plant extracts with low concentrations under lab and greenhouse conditions which started with 0.5 g/l and did not exceed 2 g/l of the dry leaves. Comparing the used concentrations with the chemical nematicides may minimize the coast effectiveness. Solidago and periwinkle might be promising candidates of nematode management tactics.

Extracts of solidago and periwinkle leaves might be promising sources of phytochemicals that have nematicidal activity.

Availability of data and materials

All data generated or analyzed in this study are available in this published manuscript.

Abbreviations

Root-knot nematode

Dry weight/liter

Gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry

Analysis of variance

Median lethal concentration

Ninety lethal concentration

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Acknowledgements

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This study was financed by Taif University Researchers Supporting Project number (TURSP-2020/92), Taif University, Taif, Saudi Arabia. This funder provided most chemicals used in plant extract and experimental bioassay.

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Zoology and Agricultural Nematology Department, Faculty of Agriculture, Cairo University, Giza, 12613, Egypt

Hosny Kesba, Abdullah Abdel-Rahman & Al-Sayed Al-Sayed

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Methodology, H.K. and A.A-R.; formal analysis, S.S. and A.A.; investigation, H.K., A.A-R, and A.A.; resources, H.K. and S.S.; data curation, A.A.; supervision, A.A. and K.K; writing—original draft preparation, H.K, A.A-R., and A.A. writing—review and editing, H.K., S.S., and A.A. All authors have read and agreed to the published version of the manuscript.

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Kesba, H., Abdel-Rahman, A., Sayed, S. et al. Screening the nematicidal potential of indigenous medicinal plant extracts against Meloidogyne incognita under lab. and greenhouse conditions. Egypt J Biol Pest Control 31 , 81 (2021). https://doi.org/10.1186/s41938-021-00429-y

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Received : 24 February 2021

Accepted : 04 May 2021

Published : 10 May 2021

DOI : https://doi.org/10.1186/s41938-021-00429-y

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• Meloidogyne incognita
• Medicinal plant extracts
• Nematicidal activity