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).

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.

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.

Clostridium difficile toxinotype XI (A-B-) exhibits unique arrangement of PaLoc and its upstream region.

Clostridium difficile toxinotype XI strains do not produce toxins A (TcdA) or B (TcdB) but do possess a nonfunctional remnant of the pathogenicity locus (PaLoc), bearing part of the sequence for tcdA. This is the first report of a type with absent upstream terminal PaLoc sequences and major genetic rearrangements of the PaLoc region.