Satellitome analyses in nematodes illuminate complex species history and show conserved features in satellite DNAs.

BACKGROUND: Satellite DNAs (satDNAs) are tandemly repeated non-coding DNA sequences that belong to the most abundant and the fastest evolving parts of the eukaryotic genome. A satellitome represents the collection of different satDNAs in a genome. Due to extreme diversity and methodological difficulties to characterize and compare satDNA collection in complex genomes, knowledge on their putative functional constraints and capacity to participate in genome evolution remains rather elusive. SatDNA transcripts have been detected in many species, however comparative studies of satDNA transcriptome between species are extremely rare. RESULTS: We conducted a genome-wide survey and comparative analyses of satellitomes among different closely related Meloidogyne spp. nematodes. The evolutionary trends of satDNAs suggest that each round of proposed polyploidization in the evolutionary history is concomitant with the addition of a new set of satDNAs in the satellitome of any particular Meloidogyne species. Successive incorporation of new sets of satDNAs in the genome along the process of polyploidization supports multiple hybridization events as the main factor responsible for the formation of these species. Through comparative analyses of 83 distinct satDNAs, we found a CENP-B box-like sequence motif conserved among 11 divergent satDNAs (similarity ranges from 36 to 74%). We also found satDNAs that harbor a splice leader (SL) sequence which, in spite of overall divergence, shows conservation across species in two putative functional regions, the 25-nt SL exon and the Sm binding site. Intra- and interspecific comparative expression analyses of the complete satDNA set in the analyzed Meloidogyne species revealed transcription profiles including a subset of 14 actively transcribed satDNAs. Among those, 9 show active transcription in every species where they are found in the genome and throughout developmental stages. CONCLUSIONS: Our results demonstrate the feasibility and power of comparative analysis of the non-coding repetitive genome for elucidation of the origin of species with a complex history. Although satDNAs generally evolve extremely quickly, the comparative analyses of 83 satDNAs detected in the analyzed Meloidogyne species revealed conserved sequence features in some satDNAs suggesting sequence evolution under selective pressure. SatDNAs that are actively transcribed in related genomes and throughout nematode development support the view that their expression is not stochastic.

First Report of the Root-Knot Nematode Meloidogyne luci on Tomato in Serbia.

Root-knot nematode (RKN) Meloidogyne luci Carneiro, Correa, Almeida, Gomes, Deimi, Castagnone-Sereno, and Karssen, 2014 was described from Brazil, Chile and Iran, parasitizing in various crops (Carneiro et al. 2014). It was later also described from Slovenia, Italy, Greece, Portugal, Turkey and Guatemala (review in Geric Stare et al. 2017). It is considered an extremely damaging pest as it has a wide host range and infects numerous higher plants, including monocotyledons and dicotyledons as well as herbaceous and woody plants. This species was included in the European Plant Protection Organisation Alert List of harmful organisms. In Europe, M. luci has been detected in both greenhouse and field agricultural production (review in Geric Stare et al. 2017). Furthermore, M. luci has been shown to survive winter in the field under continental and sub-Mediterranean climatic conditions (Strajnar et al. 2011). In August 2021, an official survey for quarantine RKN in Serbia (Province Vojvodina) revealed in a greenhouse in the village of Lugovo (43043'32,562; 19008'55,168), near Sombor, yellowing, stunning and extensive root galls on tomato (Solanum lycopersicum L.) cultivar Diva F1 caused by an unknown Meloidogyne sp. (Fig. 1). As correct identification is essential for effective pest management program, the next step was to identify the nematode species. Morphological characterization performed on freshly isolated females revealed perineal patterns similar to M. incognita (Kofoid and White, 1919) Chitwood, 1949. The shape was oval to squarish with the dorsal arch rounded to moderately high and without shoulders. The dorsal striae were wavy and continuous. The ventral striae were smooth and the lateral lines were weakly demarcated. The perivulval region was without striae (Fig. 2). The female stylet was robust with well-developed knobs and the stylet cone slightly curved dorsally. Although morphological characters was very variable, the nematode was suspected as M. luci based on comparison with originally described M. luci and M. luci populations from Slovenia, Greece and Turkey. Identification was achieved with subsequent species-specific PCR and sequence analysis. The nematode was determined to belong to the tropical RKN group and the M. ethiopica group using two PCR reactions as described by Geric Stare et al. (2019) (Figs. 3 and 4). Identification was confirmed by species-specific PCR of M. luci as described by Maleita et al. (2021), and a band of approximately 770 bp was obtained (Fig. 5). In addition, the identification was confirmed by sequence analyses. The region of mtDNA was amplified with primers C2F3 and 1108 (Powers and Harris 1993), cloned, sequenced (acc. no. OQ211107), and compared to other Meloidogyne spp. sequences from the Genbank. The determined sequence is 100% identical to an unidentified Meloidogyne sp. from Serbia, while the next highest scores are sequences of M. luci from Slovenia, Greece and Iran, all of which have 99.94% sequence identity. In phylogenetic tree, all M. luci sequences including the sequence from Serbia belong to a single clade. Egg masses isolated from infected tomato roots were used to establish a nematode culture in greenhouse and they caused typical root galls on cultivar Maraton of tomato. The galling index assessed 110 days-post-inoculation was in the range 4-5 according to the scoring scheme (1-10) for field evaluation of RKN infestations (Zeck 1971). To our knowledge, this is the first report of M. luci in Serbia. The authors hypothesize that climate change and higher temperatures could lead to much greater spread and damage to various agricultural crops in the field by M. luci in the future. National surveillance program for RKN in Serbia continued in 2022 and 2023. A management program to control the spread and damage from M. luci will be implemented in Serbia in 2023. Acknowledgments: This work was financially supported by the Serbian Plant Protection Directorate of MAFWM in the frame of Program of Measures in Plant Health in 2021, the Slovenian Research Agency in the frame of Research Programme Agrobiodiversity (P4-0072) and the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia in the frame of Expert work in the field of plant protection (C2337).

The Centromere Histone Is Conserved and Associated with Tandem Repeats Sharing a Conserved 19-bp Box in the Holocentromere of Meloidogyne Nematodes.

Although centromeres have conserved function, centromere-specific histone H3 (CenH3) and centromeric DNA evolve rapidly. The centromere drive model explains this phenomenon as a consequence of the conflict between fast-evolving DNA and CenH3, suggesting asymmetry in female meiosis as a crucial factor. We characterized evolution of the CenH3 protein in three closely related, polyploid mitotic parthenogenetic species of the Meloidogyne incognita group, and in the distantly related meiotic parthenogen Meloidogyne hapla. We identified duplication of the CenH3 gene in a putative sexual ancestral Meloidogyne. We found that one CenH3 (αCenH3) remained conserved in all extant species, including in distant Meloidogyne hapla, whereas the other evolved rapidly and under positive selection into four different CenH3 variants. This pattern of CenH3 evolution in Meloidogyne species suggests the subspecialization of CenH3s in ancestral sexual species. Immunofluorescence performed on mitotic Meloidogyne incognita revealed a dominant role of αCenH3 on its centromere, whereas the other CenH3s have lost their function in mitosis. The observed αCenH3 chromosome distribution disclosed cluster-like centromeric organization. The ChIP-Seq analysis revealed that in M. incognita αCenH3-associated DNA dominantly comprises tandem repeats, composed of divergent monomers which share a completely conserved 19-bp long box. Conserved αCenH3-associated DNA is also confirmed in the related mitotic Meloidogyne incognita group species suggesting preservation of both centromere protein and DNA constituents. We hypothesize that the absence of centromere drive in mitosis might allow for CenH3 and its associated DNA to achieve an equilibrium in which they can persist for long periods of time.

Genome sequence of the root-knot nematode Meloidogyne luci.

Root-knot nematodes from the genus Meloidogyne are polyphagous plant endoparasites and agricultural pests of global importance. Here, we report the high-quality genome sequence of Meloidogyne luci population SI-Smartno V13. The resulting genome assembly of M. luci SI-Smartno V13 consists of 327 contigs, with an N50 contig length of 1,711,905 bp and a total assembly length of 209.16 Mb.Root-knot nematodes from the genus Meloidogyne are polyphagous plant endoparasites and agricultural pests of global importance. Here, we report the high-quality genome sequence of Meloidogyne luci population SI-Smartno V13. The resulting genome assembly of M. luci SI-Smartno V13 consists of 327 contigs, with an N50 contig length of 1,711,905 bp and a total assembly length of 209.16 Mb.

Agrobacteria Enhance Plant Defense Against Root-Knot Nematodes on Tomato.

The increased incidence of the crown gall disease caused by Agrobacterium tumefaciens has long been associated with activities of root-knot nematodes (Meloidogyne spp.). Pot experiments on tomato were designed to assess plant vitality, nematode reproduction, and crown gall incidence in combined infection with Agrobacterium and Meloidogyne spp. on tomato roots. Results suggest that tomato plants infected with pathogenic A. tumefaciens 2 days before the nematodes show enhanced plant defense against M. ethiopica resulting in lower egg and gall counts on roots 45 and 90 days postinoculation (dpi); no significantly enhanced defense was observed when the plant was inoculated with bacteria and nematodes at the same time. Split-root experiments also showed that the observed interaction was systemic. Reverse-transcription quantitative polymerase chain reaction analysis that targeted several genes under plant hormonal control suggests that the suppression was mediated via systemic acquired resistance by the pathogenesis-related protein 1 and that M. ethiopica did not enhance the defense reaction of tomato against Agrobacterium spp. Nematodes completely inhibited tumor growth in a 45-day experiment if inoculated onto the roots before the pathogenic bacteria. We conclude that the observed antagonism in the tested pathosystem was the result of initially strong plant defense that was later suppressed by the invading pathogen and pest.

Reported populations of Meloidogyne ethiopica in Europe identified as Meloidogyne luci.

The tropical group of root-knot nematodes (RKN) including Meloidogyne ethiopica and M. luci is a highly polyphagus and damaging group of agricultural pests. M. ethiopica has been detected in several European countries (Slovenia, Italy, Greece) and also in Turkey. However, a description of a new sister species M. luci calls for reclassification of all European and Turkish M. ethiopica populations reported up to date as M. luci. Accurate identification can be accomplished through analysis of the esterase isozyme pattern, which is the most distinguishing character between the two otherwise very similar species. Both species display a three banded esterase pattern where the upper band is slightly shifted between the two species. In addition, molecular characterization of M. ethiopica and M. luci populations revealed that the ITS, SSU, and LSU of the rDNA regions are not appropriate markers for studying relationships among the tropical group of RKNs. However, the COII/lRNA region on mtDNA proved to be very useful for analyzing the phylogenetic relationship of these very closely related species/populations. Mitochondrial sequences with low levels of heteroplasmy allowed clustering of all M. luci populations in a monophyletic clade with a clear separation of this recently described species from M. ethiopica. At the same time, a very close relationship between M. ethiopica and M. luci was confirmed again.

Molecular variability and evolution of the pectate lyase (pel-2) parasitism gene in cyst nematodes parasitizing different solanaceous plants.

While pectate lyases are major parasitism factors in plant-parasitic nematodes, there is little information on the variability of these genes within species and their utility as pathotype or host range molecular markers. We have analysed polymorphisms of pectate lyase 2 (pel-2) gene, which degrades the unesterified polygalacturonate (pectate) of the host cell-wall, in the genus Globodera. Molecular variability of the pel-2 gene and the predicted protein was evaluated in populations of G. rostochiensis, G. pallida, G. "mexicana" and G. tabacum. Seventy eight pel-2 sequences were obtained and aligned. Point mutations were observed at 373 positions, 57% of these affect the coding part of the gene and produce 129 aa replacements. The observed polymorphism does not correlate either to the pathotypes proposed in potato cyst nematodes (PCN) or the subspecies described in tobacco cyst nematodes. The trees reveal a topology different from the admitted species topology as G. rostochiensis and G. pallida sequences are more similar to each other than to G. tabacum. Species-specific sites, potentially applicable for identification, and sites distinguishing PCN from tobacco cyst nematodes, were identified. As both G. rostochiensis and G. pallida display the same host range, but distinct from G. tabacum, which cannot parasitize potato plants, it is tempting to speculate that pel-2 genes polymorphism may be implicated in this adaptation, a view supported by the fact that no active pectate lyase 2 was found in G. "mexicana", a close relative of G. pallida that is unable to develop on cultivated potato varieties.

Evaluation of Sown Cover Crops and Spontaneous Weed Flora as a Potential Reservoir of Black-Foot Pathogens in Organic Viticulture.

(1) Background. An extensive survey of grapevine-sown cover crops and spontaneous weed flora was conducted from 2019 to 2020 in organic vineyards in six European countries (France, Italy, Romania, Slovenia, Spain, Switzerland). Our main objective was to detect and identify the presence of Cylindrocarpon-like asexual morphs species associated with black-foot disease on their roots. (2) Methods. Fungal isolations from root fragments were performed on culture media. Cylindrocarpon-like asexual morph species were identified by analyzing the DNA sequence data of the histone H3 (his3) gene region. In all, 685 plants belonging to different botanical families and genera were analyzed. Cylindrocarpon-like asexual morphs were recovered from 68 plants (9.9% of the total) and approximately 0.97% of the plated root fragments. (3) Results. Three fungal species (Dactylonectria alcacerensis, Dactylonectria torresensis, Ilyonectria robusta) were identified. Dactylonectria torresensis was the most frequent, and was isolated from many cover crop species in all six countries. A principal component analysis with the vineyard variables showed that seasonal temperatures and organic matter soil content correlated positively with Cylindrocarpon-like asexual morphs incidence. (4) Conclusions. The presence of Cylindrocarpon-like asexual morphs on roots of cover crops suggests that they can potentially act as alternative hosts for long-term survival or to increase inoculum levels in vineyard soils.

From Genome to Field-Observation of the Multimodal Nematicidal and Plant Growth-Promoting Effects of Bacillus firmus I-1582 on Tomatoes Using Hyperspectral Remote Sensing.

Root-knot nematodes are considered the most important group of plant-parasitic nematodes due to their wide range of plant hosts and subsequent role in yield losses in agricultural production systems. Chemical nematicides are the primary control method, but ecotoxicity issues with some compounds has led to their phasing-out and consequential development of new control strategies, including biological control. We evaluated the nematicidal activity of Bacillus firmus I-1582 in pot and microplot experiments against Meloidogyne luci. I-1582 reduced nematode counts by 51% and 53% compared to the untreated control in pot and microplot experiments, respectively. I-1582 presence in the rhizosphere had concurrent nematicidal and plant growth-promoting effects, measured using plant morphology, relative chlorophyll content, elemental composition and hyperspectral imaging. Hyperspectral imaging in the 400-2500 nm spectral range and supervised classification using partial least squares support vector machines successfully differentiated B. firmus-treated and untreated plants, with 97.4% and 96.3% accuracy in pot and microplot experiments, respectively. Visible and shortwave infrared spectral regions associated with chlorophyll, N-H and C-N stretches in proteins were most relevant for treatment discrimination. This study shows the ability of hyperspectral imaging to rapidly assess the success of biological measures for pest control.

Pipeline for imaging, extraction, pre-processing, and processing of time-series hyperspectral data for discriminating drought stress origin in tomatoes.

Crop infestation with root-knot nematodes (RKN) and water deficiency lead to similar visible symptoms in the plant canopy. Identification of biotic or abiotic stress origin is therefore a problem, and currently the only reliable methods for determination of RKN infestation are invasive and applicable only for point-searches. In this study the applicability of hyperspectral remote sensing for early identification of drought stress and RKN infestations in tomato plants was tested. A four-stage image and data management pipeline was established: (1) image acquisition, (2) data extraction, (3) pre-processing, and (4) processing. •This pipeline reduces atmospheric impacts, facilitates data extraction (by using specially designed spectral libraries and supervised classification procedures), diminishes the impact of viewing geometry, and emphasized small spectral variations not apparent in the raw data.•By combining partial least squares - discriminant analysis and support vector machines with time series analysis, we achieved up to 100% classification success when determining watering regime and infestation, and their severity.•This pipeline could be at least partially automated, thus facilitating high throughput identification of stress origin in plants. Furthermore, the same pipeline could be applied to hyperspectral phenotyping procedures, which are gaining importance in breeding programs.

Longidorus carniolensis sp. n. (Nematoda, Longidoridae) from vineyard soil in Slovenia.

A new needle nematode, Longidorus carniolensissp. n., recovered from the soil around the roots of grapevine Vitis vinifera L. from Slovenia, is described and illustrated. Longidorus carniolensisis an amphimictic species, characterised by females with a moderately long (L=5.6-8.2 mm) and plump (a=51-72.4, ave. 66.3) body, assuming a spiral to C-shape when heat relaxed. Head region continuous, anteriorly almost flat, lip region 23-25 µm wide; guiding ring situated posteriorly (42-47 µm, 43-50 µm in males), odontostyle long (ave. 146.6 (136-157) µm); pharyngeal glands with normal location, their nuclei of approximately equal size; tail bluntly conoidal to almost hemispherical. Males abundant, spicules slender and long (122-145 µm), ventromedian supplements 13-17, irregularly spaced, preceded by an adanal pair. Four juvenile stages present, the first stage juvenile with bluntly conoidal tail. Codes for identifying the new species when using the key by Chen et al. (1997) are: A 56, B 4, C 4, D 1, E 4, F 35, G 1, H 1, I 2. The new species is morphologically the most similar to Longidorus poessneckensis Altherr, 1974, Longidorus macrosoma Hooper, 1961, Longidorus caespiticola Hooper, 1961, Longidorus helveticus Lamberti et al., 2001, Longidorus macroteromucronatus Altherr, 1974, Longidorus pius Barsi & Lamberti, 2001, Longidorus raskii Lamberti & Agostinelli, 1993, Longidorus kheirii Pedram et al. 2008, Longidorus silvae Roca, 1993, Longidorus iuglandis Roca et al., 1985, Longidorus vinearum Bravo & Roca, 1995 and Longidorus major Roca & d'Erico, 1987, but differs from these species either by the body and odontostyle length, position of guide ring, head region and tail shape or the shape of the first stage juvenile tail. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other speciesof the genus Longidorus with known sequences. Relationships of Longidorus carniolensissp. n. with other Longidorus species based on analysis of this DNA fragment and morphology are discussed.