An early record of Meloidogyne fallax from Ireland

Root-knot nematodes, Meloidogyne spp., cause huge economic losses worldwide. Currently, three Meloidogyne spp. are present on the quarantine A2 list of EPPO, M. chitwoodi, M. fallax and M. enterolobii. As a quarantine organism, M. fallax has been detected in England and Northern Ireland on sport turf in 2011, and in England on leek in 2013. However, its presence in Ireland has probably been overlooked since 1965, when Mr. John F. Moore and Dr. Mary T. Franklin had detected a new Meloidogyne species for that time. While the relevant data was recorded and a preliminary manuscript describing the species was prepared but never submitted for publication, and together with the original slides, pictures and drawings, it was restudied recently. We compared the population of Irish Meloidogyne sp. to other similar Meloidogyne spp. Careful observation and comparison shows that it belongs to M. fallax. The characters found to be common for Irish Meloidogyne sp. and M. fallax are female stylet length (14.6 mu m) with oval to rounded basal knobs, oval shaped perineal pattern with moderately high dorsal arch, slender stylet in males (18.5 mu m) with set off and rounded basal knobs, slightly set off male head with one post-labial annule and incomplete transverse incisures, and second-stage juveniles with large and rounded stylet basal knobs, and a gradually tapering tail (46.9 mu m) with a broadly rounded tip and a clearly delimitated smooth hyaline part sometimes marked by constrictions (12.9 mu m). The host test and gall formation also correspond to M. fallax. The identification could not be additionally supported by molecular analysis, as we were unable to extract DNA from the old permanent slides. Nevertheless, our study reveals that the Meloidogyne species detected in Ireland in 1965 belongs to M. fallax

Clofazimine acid-base solubilization: influence of small organic acids’ concentration

Methods for drug solubilization have become important part of modern drug discovery and development due to increasing number of extremely insoluble drugs and drug candidates. One of such methods is acid-base supersolubilization (ABS) [1]. Clofazimine (CFZ) is weakly basic antibiotic and anti-inflammatory drug, most notably used in the treatment of leprosy and tuberculosis, with recently proven inhibitory activity against several coronaviruses [2]. We have recently unraveled its aqueous pKa value and its unique cosolvent dependence [3]. The aim of the present study was to investigate CFZ solubilization using the ABS approach. Eight small organic acids were tested for the ABS effect (glutaric, malic, tartaric, citric, malonic, maleic, succinic, adipic) but only glutaric (GA), malic (MA), and tartaric (TA) acids showed some solubilization effect. The effect of their concentration (and the solution pH value) was further tested. The solubility of CFZ was determined in GA, MA, and TA solutions in wide concentration (1.0×10-2 – 5.0 M) and pH range (~0.2 – 4.8). Equilibration time was 24 hours (6 h of stirring + 18 h of sedimentation). Phases were separated by filtration. The CFZ concentration in supernatant was determined by HPLC-UV/VIS. Results show that CFZ solubility increases as acid concentration increases: from 3.04×10-3 to 10.68 mg/mL (in GA), from 9.06×10-3 to 1.23 mg/mL (in MA) and from 4.76×10-3 to 0.32 mg/mL (in TA). The effect of CFZ solubilization is much more pronounced when the acid concentration is raised above 2 M. These results can be used as the basis for further CFZ formulation optimization. Furthermore, our ongoing research is focused on the type of interactions and other possible factors that can influence CFZ and other prectically insoluble drugs, embracing (super)solubilization as a general methodology in drug design and development

Clofazimine acid-base solubilization: influence of small organic acids’ concentration

Methods for drug solubilization have become important part of modern drug discovery and development due to increasing number of extremely insoluble drugs and drug candidates. One of such methods is acid-base supersolubilization (ABS) [1]. Clofazimine (CFZ) is weakly basic antibiotic and anti-inflammatory drug, most notably used in the treatment of leprosy and tuberculosis, with recently proven inhibitory activity against several coronaviruses [2]. We have recently unraveled its aqueous pKa value and its unique cosolvent dependence [3]. The aim of the present study was to investigate CFZ solubilization using the ABS approach. Eight small organic acids were tested for the ABS effect (glutaric, malic, tartaric, citric, malonic, maleic, succinic, adipic) but only glutaric (GA), malic (MA), and tartaric (TA) acids showed some solubilization effect. The effect of their concentration (and the solution pH value) was further tested. The solubility of CFZ was determined in GA, MA, and TA solutions in wide concentration (1.0×10-2 – 5.0 M) and pH range (~0.2 – 4.8). Equilibration time was 24 hours (6 h of stirring + 18 h of sedimentation). Phases were separated by filtration. The CFZ concentration in supernatant was determined by HPLC-UV/VIS. Results show that CFZ solubility increases as acid concentration increases: from 3.04×10-3 to 10.68 mg/mL (in GA), from 9.06×10-3 to 1.23 mg/mL (in MA) and from 4.76×10-3 to 0.32 mg/mL (in TA). The effect of CFZ solubilization is much more pronounced when the acid concentration is raised above 2 M. These results can be used as the basis for further CFZ formulation optimization. Furthermore, our ongoing research is focused on the type of interactions and other possible factors that can influence CFZ and other prectically insoluble drugs, embracing (super)solu bilization as a general methodology in drug design and development

Revealing the story of an orphan drug: clofazimine speciation and solubilization as a function of pH

Since the introduction of combinatorial chemistry and high-throughput screening in drug discovery in the early 1990s, the solubility of new chemical entities (NCE) decreased drastically while their lipophilicities increased greatly. Characterizing physicochemical properties of low soluble molecules can be especially challenging, since such molecules can undergo complicated reactions in aqueous solution, such as forming precipitates or complexes with buffer species or undergoing self-aggregation (dimer, trimer, etc.) or micelle formations. Most drugs are ionizable. Foremost to the rational interpretation of solution behavior of ionizable drugs in a physiologically-relevant pH domain requires an accurate aqueous pKa, determined by a suitable method. In a pH-dependent measurement of a property (e.g. solubility-, lipophilicity-, permeability-pH), when the apparent pKa value is different from the true aqueous pKa value, it may be an early clue that nonideal solution behavior may be taking place. In pharmaceutical research, it may seem cost-effective to use calculated pKa instead of measured values, but paradoxically, such preference can lead to inaccurate rationalization of the pH-dependent behavior of the drug molecule. For simple molecules, calculated values can be useful, but for today’s new drugs or for molecules prone to complicated solution behavior, the use of calculated pKas can substantially wrench the interpretation of solution properties. Clofazimine (CFZ), although discovered about 66 years ago, and used therapeutically for nearly 40 years, exhibits some of the properties of relatively recent drug molecules by being extremely water insoluble and having variable pKa values reported. We have recently combined potentiometric titrations and UV/Vis spectrophotometry in methanol-water cosolvent media, accompanied by DFT calculations, to assess the hypothesis of CFZ free base dimerization. We reasoned that a soluble dimer might form from drug-drug adhesion along the hydrophobic molecular surface. With lessened exposure of the hydrophobic surface to water, the dimer would be more water soluble than the monomeric free base. In saturated solutions, the apparent solubility in alkaline pH would be elevated due to the presence of the dimer. The effect of that would be a lower pKa and reverse pKa cosolvent dependence – the behaviour we have noticed in CFZ aqueous solutions. These findings are of paramount importance for understanding of CFZ speciation and the future progress in developing its improved formulations which is the subject of our ongoing studies

Plant-nematode interactions assisted by microbes in the rhizosphere

Plant health is strongly influenced by the interactions between parasites/pathogens and beneficial microorganisms. In this chapter we will summarize the up-to date knowledge on soil suppressiveness as a biological tool against phytonematodes and explore the nature of monoculture versus crop rotation in this regard. Since nematodes are successfully antagonized by different microbiological agents, we highlighted this phenomenon with respect to the most important antagonists, and a nature of these interactions. The focus is on the hyperparasitic microbes of phytonematodes such as Pasteuria sp. and egg parasites. Furthermore, we comprised the studies on the defence system expressions in plants triggered by nematode-associated microbes. The attachment of bacteria and fungi to phytonematodes and putative effects of the attachment on the induced systemic resistance in plants are discussed. Finally, our chapter is rounded up with the importance of incorporating the knowledge on plant-nematode-microbe interactions in the integrated pest management

Plants specifically modulate the microbiome of root-lesion nematodes in the rhizosphere, affecting their fitness

Plant-parasitic nematodes are a major constraint on agricultural production. They significantly impede crop yield. To complete their parasitism, they need to locate, disguise, and interact with plant signals exuded in the rhizosphere of the host plant. A specific subset of the soil microbiome can attach to the surface of nematodes in a specific manner. We hypothesized that host plants recruit species of microbes as helpers against attacking nematode species, and that these helpers differ among plant species. We investigated to what extend the attached microbial species are determined by plant species, their root exudates, and how these microbes affect nematodes. We conditioned the soil microbiome in the rhizosphere of different plant species, then employed culture-independent and culture-dependent methods to study microbial attachment to the cuticle of the phytonematode Pratylenchus penetrans. Community fingerprints of nematode-attached fungi and bacteria showed that the plant species govern the microbiome associated with the nematode cuticle. Bacteria isolated from the cuticle belonged to Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Sphingobacteria, and Firmicutes. The isolates Microbacterium sp. i.14, Lysobacter capsici i.17, and Alcaligenes sp. i.37 showed the highest attachment rates to the cuticle. The isolates Bacillus cereus i.24 and L. capsici i.17 significantly antagonized P. penetrans after attachment. Significantly more bacteria attached to P. penetrans in microbiome suspensions from bulk soil or oat rhizosphere compared to Ethiopian mustard rhizosphere. However, the latter caused a better suppression of the nematode. Conditioning the cuticle of P. penetrans with root exudates significantly decreased the number of Microbacterium sp. i.14 attaching to the cuticle, suggesting induced changes of the cuticle structure. These findings will lead to a more knowledge-driven exploitation of microbial antagonists of plant-parasitic nematodes for plant protection

Plants and Associated Soil Microbiota Cooperatively Suppress Plant-Parasitic Nematodes

Disease suppressive soils with specific suppression of soil-borne pathogens and parasites have been long studied and are most often of microbiological origin. As for the plant-parasitic nematodes (PPN), which represent a huge threat to agricultural crops and which successfully defy many conventional control methods, soil progression from conducive to suppressive state is accompanied by the enrichment of specific antagonistic microbial consortia. However, a few microbial groups have come to the fore in diminishing PPN in disease suppressive soils using culture-dependent methods. Studies with cultured strains resulted in understanding the mechanisms by which nematodes are antagonized by microorganisms. Recent culture-independent studies on the microbiome associated with soil, plant roots, and PPN contributed to a better understanding of the functional potential of disease suppressive microbial cohort. Plant root exudation is an important pathway determining host-microbe communication and plays a key role in selection and enrichment of a specific set of microbial antagonists in the rhizosphere as first line of defense against crop pathogens or parasites. Root exudates comprising primary metabolites such as amino acids, sugars, organic acids, and secondary metabolites can also cause modifications in the nematode surface and subsequently affect microbial attachment. A positive interaction between hosts and their beneficial root microbiota is correlated with a low nematode performance on the host. In this review, we first summarized the historical records of nematode-suppressive soils and then focused on more recent studies in this aspect, emphasizing the advances in studying nematode-microbe interactions over time. We highlighted nematode biocontrol mechanisms, especially parasitism, induced systemic resistance, and volatile organic compounds using microbial consortia, or bacterial strains of the genera Pasteuria, Bacillus, Pseudomonas, Rhizobium, Streptomyces, Arthrobacter, and Variovorax, or fungal isolates of Pochonia, Dactylella, Nematophthora, Purpureocillium, Trichoderma, Hirsutella, Arthrobotrys, and Mortierella. We discussed the importance of root exudates in plant communication with PPN and soil microorganisms, emphasizing their role in microbial attachment to the nematode surface and subsequent events of nematode parasitism. Comprehensive understanding of the plant-beneficial microbial consortia and the mechanisms underlying disease suppression may help to develop synthetic microbial communities for biocontrol of PPN, thereby reducing nematicides and fertilizers inputs

Microbes Attaching to Endoparasitic Phytonematodes in Soil Trigger Plant Defense Upon Root Penetration by the Nematode

Root-knot nematodes (Meloidogyne spp.) are among the most aggressive phytonematodes. While moving through soil to reach the roots of their host, specific microbes attach to the cuticle of the infective second-stage juveniles (J2). Reportedly, the attached microorganisms affect nematodes and reduce their performance on the host plants. We have previously shown that some non-parasitic bacterial strains isolated from the cuticle of Meloidogyne hapla in different soils affected J2 mortality, motility, hatching, and root invasion. Here we tested whether cuticle-attached microbes trigger plant defenses upon penetration of J2. In in vitro assays, M. hapla J2-attached microbes from a suppressive soil induced pathogen-associated molecular pattern-triggered immunity (PTI) in tomato roots. All tested PTI-responsive defense genes were upregulated after root invasion of J2 with attached microbes, compared to surface-sterilized J2, particularly the jasmonic acid-mediated PTI marker genes TFT1 and GRAS4.1. The strain Microbacterium sp. K6, that was isolated from the cuticle, significantly reduced root invasion when attached to the J2. Attached K6 cells supported plant defense and counteracted suppression of plant basal defense in roots by invaded J2. The plant response to the J2-attached K6 cells was stronger in leaves than in roots, and it increased from 1 to 3 days post inoculation (dpi). At 1 dpi, the plant responded to J2-attached K6 cells by ameliorating the J2-triggered down-regulation of defense genes mostly in roots, while at 3 dpi this response was systemic and more pronounced in leaves. In a reactive oxygen species (ROS) assay, the compounds released from J2 with attached K6 cells triggered a stronger ROS burst in tomato roots than the compounds from nematodes without K6, or the metabolites released from strain K6 alone. Leaves showed a 100 times more sensitive response than roots, and the metabolites of K6 with or without J2 induced strong ROS bursts. In conclusion, our results suggest the importance of microorganisms that attach to M. hapla in suppressive soil, inducing early basal defenses in plants and suppressing nematode performance in roots