Analysing nematode behaviour helps estimate biomechanical parameters for applications like cellular biology, pharmacology and cognitive neuroscience. Portable holographic platforms offer cost-effective, high-resolution, high-frame-rate, wide-field imaging compared to conventional microscopy. Holographic methods can reconstruct original shapes using numerical diffraction, although this is computationally expensive. However, video holography remains challenging due to the fast motion and overlapping of holograms when nematodes swim in crowded environments. In this work we address this problem by focusing on automated detection and tracking of nematodes in densely populated environments, using machine learning methods. The main advantage of our approach is to present an automated computational flow to detect and analyse the behaviour of live nematodes in video directly from the raw holographic signals, without the requirement of phase-recovering methods for diffraction. For this purpose, we developed a three-step CNN-based approach consisting of: i) nematode hologram detection; ii) temporal tracking; and iii) behavioural analysis based on mobility parameters. In terms of precision, the obtained results show that using a two-stage detector, the Faster R-CNN architecture with the ResNet18 model as a backbone, presented the best Mean Average Precision (mAP) score with 86% for correct classification. For tracking, the best performing algorithm was IoU with a HOTA, resulting in 62.42% when applied on the individually tagged nematodes, which is comparable to the best current generic multi-tracking approaches available over the literature. Our obtained results show that the use of a convolutional neural network approach associated with a classic tracking algorithm is a very suitable approach for nematode detection and behavioural analysis for biological assays directly from holograms, even in densely populated environments. The proposed approach has been presented as a promising solution for automated inspection of free-living nematode individuals.
The use of non-host cover crops can be a viable strategy for managing soybean cyst nematode (SCN) Heterodera glycines. Cover crops may affect SCN biology and reduce populations by acting as trap crops that promote the hatching and/or penetration by second-stage juveniles (J2) without supporting their development. Two growth chamber experiments were conducted to assess ten cover crops for their impacts on SCN hatching and penetration into the roots. For each experiment, with four replications per treatment, crops were planted in naturally-infested soil in two separate sets to be harvested 15 and 30 days after planting (DAP). SCN susceptible soybean ‘Barnes’ and non-planted natural soil (fallow) were used as controls. Faba bean ‘Petite’, a non-host of SCN, induced the greatest hatching among the cover crops and was statistically similar to soybean. Winter rye ‘ND Dylan’ also induced significant hatching compared to fallow. Root staining revealed that the highest number of J2 penetrated the faba bean roots at 15 DAP, followed by soybean and winter rye. While J2 penetrated all tested crops, they completed their development to become adult females only in soybean and turnip. Soybean cyst nematode development to adult females did not occur in faba bean, and the number of SCN inside the faba bean roots 30 DAP was significantly lower than at 15 DAP and also lower than in soybean. These results suggest that the faba bean affects SCN biology and has the greatest potential to act as a trap crop for managing SCN.
Limited shelf life is a major constraint to successful commercialisation of entomopathogenic nematodes (EPN), and to extend shelf life, dauer juveniles (DJ) are formulated and stored at low temperatures (4-8°C). We evaluated the cold storage potential of strains of Heterorhabditis bacteriophora formulated in diatomaceous earth at storage temperatures between 5 and 9°C. When assessing DJ decline to reach 75% survival (MT75) in the formulation for the respective temperatures, H. bacteriophora strain HB4 had the highest survival of 25 days at 9°C, while strain D2D6 survived longest at 8°C for 28 days. A set of 22 H. bacteriophora wild type inbred lines was then phenotyped for cold tolerance in water under oxidative stress in 70 mM H2O2 at 2°C. The MT50 (time to 50% survival) ranged from 11 to 23 days. The phenotypic data were correlated with the respective genotypic data, identifying four single nucleotide polymorphic (SNP) markers associated with cold tolerance. The survival of two lines (PT11 and IR11) with opposite extreme cold tolerance pheno- and genotypes was evaluated in diatomaceous earth formulation at 2°C with the cold tolerant IR11 surviving 3 days longer than PT11. Our study yields a set of valuable SNP markers employable in rapid genotyping of cold tolerance and tracking this trait during the breeding process.
Rotylenchus astragalus sp. n. is characterised by having females 841-1069 μm long, stylet 28-31 μm long, basal knobs rounded, dorsal pharyngeal gland orifice (DGO) 3-5 μm, lateral field with four incisures, pharyngeal gland lobes slender, overlapping intestine dorsally for 4-12 μm, excretory pore located just posterior to the hemizonid, vulva slightly posterior to mid-body, ovaries paired, phasmid opening relatively large and 8-18 annuli anterior to the anus, tail conoid and absence of males. The matrix code of R. astragalus sp. n. is A5, B3, C1, D4, E1, F2, G1-2, H3, I2, J2, K1. In addition, phylogenetic relationships of the recovered populations were analysed using sequences of the near full length small subunit (SSU) and D2-D3 segments of large subunit (LSU) rRNA genes.
Bush tea (Athrixia phylicoides) is an aromatic, perennial, leafy shrub that is endemic to the northeastern mountain ranges of South Africa and has a high potential for commercialisation as an alternative to caffeine-containing tea. During the summer and winter of 2018-2019, a survey was carried out at nine localities in the northeastern regions of South Africa to study the diversity of nematodes associated with bush tea and, in terms of frequency of occurrence and abundance, identify the dominant plant-parasitic nematodes. Twenty-one plant-parasitic nematode species belonging to 14 genera were identified in 90 rhizosphere soil and root samples. Meloidogyne and Helicotylenchus were the dominant plant-parasitic nematode genera. Meloidogyne javanica and Scutellonema brachyurus were found at all localities, followed by M. enterolobii, Pratylenchus brachyurus, Rotylenchulus parvus, H. martini and S. truncatum (found at 7-8 localities). Other species identified included Criconema corbetii, C. sphaerocephalus, C. xenoplax, Criconemoides ihlathum, C. parvus, Discocriconemella glabrannulata, H. dihystera, H. erythrinae, H. paraplatyurus, Hemicycliophora typica, M. hapla, M. incognita, Rotylenchulus unisexus and R. clavicaudatus. Individuals of Crossonema, Paratylenchus, Ogma, the Xiphinema americanum-group and X. americanum sensu lato could not be identified to species level due to the low number of specimens present in the samples. Comparison of the two methods used to identify the Meloidogyne populations to species level shows that morphological identification (particularly perineal pattern morphology) provided a more complete picture of the Meloidogyne species present in the samples compared with the molecular SCAR-PCR technique. High levels of Cu, K and pH were associated with the highest relative population densities (RPD% = average population density of a nematode genus/total nematode population density × 100) of Meloidogyne, whilst high levels of Al and soil resistivity were associated with the lowest RPD% of Meloidogyne. By contrast, high levels of K and pH were associated with the lowest RPD% of Helicotylenchus, whilst high levels of Al and soil resistivity were associated with the highest RPD% of Helicotylenchus.