First report of infections with isolates of the Asian genotype of Cucumber green mottle mosaic virus in cucumber crops in Belgium.

In July 2019, four rows of cucumber plants (Cucumis sativus) in a commercial glasshouse in the north of Belgium showed severe mosaic, blistering and distortion of the leaves, with symptoms resembling those caused by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a Tobamovirus that mainly affects cucurbit crops worldwide (Dombrovsky et al., 2017). Phylogenetic analyses in previous studies have shown two major clades, one including isolates that were initially identified in Europe and Russia (European genotype) and the second one with isolates initially identified in Asia and Israel (Asian genotype) (Dombrovsky et al., 2017; Pitman et al., 2022; Mackie et al., 2023). A symptomatic leaf sample was collected and total RNA was isolated from 100 mg of leaf tissue (Spectrum™ Plant Total RNA kit, Sigma-Aldrich). CGMMV was detected using a one-step TaqMan RT-qPCR (Hongyun et al., 2008). High-throughput sequencing (HTS) confirmed the presence of CGMMV. The sample was prepared using the Novel enrichment technique of viromes protocol (NETOVIR protocol, Conceição-Neto et al., 2015). The leaf material was homogenized, enriched for virus-like particles and the RNA was extracted (QIAamp Viral RNA mini kit, QIAGEN). The extract was randomly amplified (Whole Transcriptome Amplification kit, Sigma Aldrich), used for library preparation (Nextera XT DNA library preparation kit, Illumina) and sequenced on a NovaSeq platform. HTS data analysis was performed using Geneious Prime software (Biomatters, Auckland, New Zealand, version 2023.2). After quality filtering and trimming, 26.7M reads were obtained (132 nt mean length). In total, 20.6M reads were mapped to two genomes KP772568 and GQ411361 (considered as reference for the Asian and European genotypes respectively) with Geneious. This revealed 100% coverage of the full sequences (6422 nt) with 99.4% and 90% nucleotide identities to the reference genomes of Asian and European genotypes, respectively. Phylogenetic analyses confirmed that isolate 2019-26A-BE, with GenBank ID OR724740, relates to the Asian genotype. The HTS data were additionally processed using the ViPER pipeline (De Coninck, 2021). The raw reads were quality filtered and trimmed, (Trimmomatic) and then used to perform de novo assembly (metaSPAdes). The produced contigs were classified using DIAMOND and visualized with KronaTools. The results showed that no other virus was detected in the sample. Finally, cucumber seedlings were inoculated using the original symptomatic sample and were grown in a research glasshouse. After 3 weeks, severe CGMMV symptoms, similar to the original symptoms observed in the commercial glasshouse, were observed in the inoculated plants. Infection with CGMMV was verified via RT-qPCR, and the isolate present in the inoculated plants was confirmed to belong to the Asian genotype via RT-PCR-RFLP (Crespo et al., 2017). Later samplings of symptomatic leaves confirmed the presence of isolates belonging to the Asian genotype of CGMMV at four other commercial glasshouse locations, specializing in cucumber crop, in Belgium in 2020, 2021 and 2023 by RT-PCR-RFLP. While the Asian genotype was previously found elsewhere in Europe (Pitman et al., 2022), to our knowledge, this is the first report of infections with isolates of this genotype in glasshouse cucumber crops in Belgium. Further investigation is required to determine the spread and impact of infections with isolates of the Asian genotype in cucumber crops in Belgium.

Single amino acid change in tomato brown rugose fruit virus breaks virus-specific resistance in new resistant tomato cultivar.

Introduction: Tomato cultivation across the world is severely affected by emerging plant viruses. An effective method for protection of commercial crops against viral threats is the use of cultivars harboring resistance genes. Tomato brown rugose fruit virus (ToBRFV), a recently emerged tobamovirus, is able to overcome the dominant Tm-22 resistance that is present in the majority of commercial tomato cultivars. In an effort to alleviate the severe consequences of ToBRFV on tomato production, tomato breeding companies are developing new cultivars with varying levels of resistance against ToBRFV. Methods: In the present study, cultivars with a new resistant phenotype against ToBRFV were screened against a wild-type isolate of ToBRFV, and subsequently, their performance under commercial greenhouse conditions was monitored. Following the identification of ToBRFV symptoms in a commercial greenhouse-where both new resistant and susceptible cultivars were interplanted-these cultivars were more closely examined. Results: The presence of ToBRFV was molecularly confirmed on both cultivar types suggesting that the new resistance had been broken. High-throughput sequencing (HTS) was used to study the complete genomes of viral isolates present in the two cultivar types. The analysis revealed a single amino acid change at position 82 of the movement protein of ToBRFV in the isolate present in the new resistant cultivar compared with the isolate identified in the susceptible cultivar. Discussion: A screening bioassay, that was performed to compare the infectivity of the two ToBRFV isolates, confirmed that only the isolate with this specific amino acid change could successfully infect the resistant cultivar, overcoming the new resistance against ToBRFV.

RNAseq-based transcriptome analysis of Lactuca sativa infected by the fungal necrotroph Botrytis cinerea.

The fungal pathogen Botrytis cinerea establishes a necrotrophic interaction with its host plants, including lettuce (Lactuca sativa), causing it to wilt, collapse and eventually dry up and die, which results in serious economic losses. Global expression profiling using RNAseq and the newly sequenced lettuce genome identified a complex network of genes involved in the lettuce-B. cinerea interaction. The observed high number of differentially expressed genes allowed us to classify them according to the biological pathways in which they are implicated, generating a holistic picture. Most pronounced were the induction of the phenylpropanoid pathway and terpenoid biosynthesis, whereas photosynthesis was globally down-regulated at 48 h post-inoculation. Large-scale comparison with data available on the interaction of B. cinerea with the model plant Arabidopsis thaliana revealed both general and species-specific responses to infection with this pathogen. Surprisingly, expression analysis of selected genes could not detect significant systemic transcriptional alterations in lettuce leaves distant from the inoculation site. Additionally, we assessed the response of these lettuce genes to a biotrophic pathogen, Bremia lactucae, revealing that similar pathways are induced during compatible interactions of lettuce with necrotrophic and biotrophic pathogens.

Mining the genome of Arabidopsis thaliana as a basis for the identification of novel bioactive peptides involved in oxidative stress tolerance.

Although evidence has accumulated on the role of plant peptides in the response to external conditions, the number of peptide-encoding genes in the genome is still underestimated. Using tiling arrays, we identified 176 unannotated transcriptionally active regions (TARs) in Arabidopsis thaliana that were induced upon oxidative stress generated by the herbicide paraquat (PQ). These 176 TARs could be translated into 575 putative oxidative stress-induced peptides (OSIPs). A high-throughput functional assay was used in the eukaryotic model organism Saccharomyces cerevisiae allowing us to test for bioactive peptides that increase oxidative stress tolerance. In this way, we identified three OSIPs that, upon overexpression in yeast, resulted in a significant rise in tolerance to hydrogen peroxide (H2O2). For one of these peptides, the decapeptide OSIP108, exogenous application to H2O2-treated yeast also resulted in significantly increased survival. OSIP108 is contained within a pseudogene and is induced in A. thaliana leaves by both the reactive oxygen species-inducer PQ and the necrotrophic fungal pathogen Botrytis cinerea. Moreover, infiltration and overexpression of OSIP108 in A. thaliana leaves resulted in increased tolerance to treatment with PQ. In conclusion, the identification and characterization of OSIP108 confirms the validity of our high-throughput approach, based on tiling array analysis in A. thaliana and functional screening in yeast, to identify bioactive peptides.

Effects of Rhizophagus irregularis MUCL 41833 on the reproduction of Radopholus similis in banana plantlets grown under in vitro culture conditions.

The role of arbuscular mycorrhizal fungi (AMF) in the control of migratory endoparasitic nematodes is nowadays largely admitted. Most studies were conducted under greenhouse conditions and a few used in vitro cultures with transgenic root organs. Here, we reported, for the first time, on the interaction between an AMF, Rhizophagus irregularis MUCL 41833 and Radopholus similis in roots of banana plantlets grown under in vitro culture conditions. The banana plantlets were pre-mycorrhized in an extraradical mycelium network arising from a Medicago truncatula donor seedling, before transfer to an autotrophic in vitro cultivation system and subsequent nematode inoculation. Both microorganisms were able to complete their life cycle in the absence as well as in presence of each other. The total R. similis population (i.e., summed over the roots and growth medium) as well as the surface of root necrosis was significantly reduced by 60 and 56 %, respectively, in the AMF-colonized banana plantlets. By contrast, nematodes had no visible impact on root colonization (i.e., percentage of arbuscules, intraradical spores/vesicles, and hyphae) by AMF and on the number of spores and hyphal length produced in the medium. These results clearly demonstrated that pre-mycorrhized banana plants could outcompete R. similis, while root colonization was not affected by the nematodes. They underline the interest of the novel in vitro cultivation system as a promising tool to investigate the biochemical factors and molecular mechanisms involved in the bio-protection conferred by AMF to a major root pathogen of banana.

Tomato brown rugose fruit virus in aqueous environments - survival and significance of water-mediated transmission.

Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control.

Arbuscular mycorrhizal fungi affect both penetration and further life stage development of root-knot nematodes in tomato.

The root-knot nematode Meloidogyne incognita poses a worldwide threat to agriculture, with an increasing demand for alternative control options since most common nematicides are being withdrawn due to environmental concerns. The biocontrol potential of arbuscular mycorrhizal fungi (AMF) against plant-parasitic nematodes has been demonstrated, but the modes of action remain to be unraveled. In this study, M. incognita penetration of second-stage juveniles at 4, 8 and 12 days after inoculation was compared in tomato roots (Solanum lycopersicum cv. Marmande) pre-colonized or not by the AMF Glomus mosseae. Further life stage development of the juveniles was also observed in both control and mycorrhizal roots at 12 days, 3 weeks and 4 weeks after inoculation by means of acid fuchsin staining. Penetration was significantly lower in mycorrhizal roots, with a reduction up to 32%. Significantly lower numbers of third- and fourth-stage juveniles and females accumulated in mycorrhizal roots, at a slower rate than in control roots. The results show for the first time that G. mosseae continuously suppresses root-knot nematodes throughout their entire early infection phase of root penetration and subsequent life stage development.

Non-interventional study comparing treatment satisfaction in patients treated with antihistamines.

BACKGROUND AND OBJECTIVE: Allergic rhinitis and urticaria are common allergic disorders that may affect approximately 15% of people at some time in their lives. Antihistamines are the most widely used therapeutic interventions for these disorders but the newer generation agents have differing pharmacokinetic characteristics that may result in different patient satisfaction and preferences. The objective of this study was to investigate patients' and physicians' satisfaction with their current antihistamine treatment for allergic disease. METHODS: In an observational study, physicians in nine European countries completed questionnaires evaluating 7,274 patients treated with an oral antihistamine. The satisfaction of patients and physicians with the efficacy and tolerability of treatment was rated on a visual analogue scale. In addition, the proportion of patients satisfied with treatment (overall satisfaction) and willing to continue treatment with the same antihistamine were assessed. Safety and tolerability data were also gathered. RESULTS: The results of this study indicate that modern antihistamines are generally considered effective and well tolerated by patients. In general, levocetirizine scored significantly higher in terms of perception of efficacy, tolerability and overall satisfaction. In terms of tolerability, three-quarters of patients were 'very satisfied' and a further fifth were moderately satisfied with levocetirizine and almost all (95%) were happy to continue treatment. Overall, the most commonly reported adverse event in this study was somnolence, a well known effect of antihistamines. The rate of somnolence in the levocetirizine group (3.8%) was similar to that for fexofenadine (both doses) and desloratadine, two products which are considered to be nonsedating antihistamines, and significantly less than half the rate for cetirizine. CONCLUSION: Levocetirizine is considered an effective and well tolerated option for treating allergic disease by patients and physicians alike, particularly when the best available effectiveness and tolerability are required.

Fungal Glucosylceramide-Specific Camelid Single Domain Antibodies Are Characterized by Broad Spectrum Antifungal Activity.

Chemical crop protection is widely used to control plant diseases. However, the adverse effects of pesticide use on human health and environment, resistance development and the impact of regulatory requirements on the crop protection market urges the agrochemical industry to explore innovative and alternative approaches. In that context, we demonstrate here the potential of camelid single domain antibodies (VHHs) generated against fungal glucosylceramides (fGlcCer), important pathogenicity factors. To this end, llamas were immunized with purified fGlcCer and a mixture of mycelium and spores of the fungus Botrytis cinerea, one of the most important plant pathogenic fungi. The llama immune repertoire was subsequently cloned in a phage display vector to generate a library with a diversity of at least 108 different clones. This library was incubated with fGlcCer to identify phages that bind to fGlcCer, and VHHs that specifically bound fGlcCer but not mammalian or plant-derived GlcCer were selected. They were shown to inhibit the growth of B. cinerea in vitro, with VHH 41D01 having the highest antifungal activity. Moreover, VHH 41D01 could reduce disease symptoms induced by B. cinerea when sprayed on tomato leaves. Based on all these data, anti-fGlcCer VHHs show the potential to be used as an alternative approach to combat fungal plant diseases.

Arbuscular Mycorrhizal Fungi for the Biocontrol of Plant-Parasitic Nematodes: A Review of the Mechanisms Involved.

Arbuscular mycorrhizal fungi (AMF) are obligate root symbionts that can protect their host plant against biotic stress factors such as plant-parasitic nematode (PPN) infection. PPN consist of a wide range of species with different life styles that can cause major damage in many important crops worldwide. Various mechanisms have been proposed to play a role in the biocontrol effect of AMF against PPN. This review presents an overview of the different mechanisms that have been proposed, and discusses into more detail the plausibility of their involvement in the biocontrol against PPN specifically. The proposed mechanisms include enhanced plant tolerance, direct competition for nutrients and space, induced systemic resistance (ISR) and altered rhizosphere interactions. Recent studies have emphasized the importance of ISR in biocontrol and are increasingly placing rhizosphere effects on the foreground as well, both of which will be the focal point of this review. Though AMF are not yet widely used in conventional agriculture, recent data help to develop a better insight into the modes of action, which will eventually lead toward future field applications of AMF against PPN. The scientific community has entered an exciting era that provides the tools to actually unravel the underlying molecular mechanisms, making this a timely opportunity for a review of our current knowledge and the challenges ahead.

What lies beneath: belowground defense strategies in plants.

Diseases caused by soil-borne pathogens result worldwide in significant yield losses in economically important crops. In contrast to foliar diseases, relatively little is known about the nature of root defenses against these pathogens. This review summarizes the current knowledge on root infection strategies, root-specific preformed barriers, pathogen recognition, and defense signaling. Studies reviewed here suggest that many commonalities as well as differences exist in defense strategies employed by roots and foliar tissues during pathogen attack. Importantly, in addition to pathogens, plant roots interact with a plethora of non-pathogenic and symbiotic microorganisms. Therefore, a good understanding of how plant roots interact with the microbiome would be particularly important to engineer resistance to root pathogens without negatively altering root-beneficial microbe interactions.

The toolbox of Trichoderma spp. in the biocontrol of Botrytis cinerea disease.

Botrytis cinerea is a necrotrophic fungal pathogen causing disease in many plant species, leading to economically important crop losses. So far, fungicides have been widely used to control this pathogen. However, in addition to their detrimental effects on the environment and potential risks for human health, increasing fungicide resistance has been observed in the B. cinerea population. Biological control, that is the application of microbial organisms to reduce disease, has gained importance as an alternative or complementary approach to fungicides. In this respect, the genus Trichoderma constitutes a promising pool of organisms with potential for B. cinerea control. In the first part of this article, we review the specific mechanisms involved in the direct interaction between the two fungi, including mycoparasitism, the production of antimicrobial compounds and enzymes (collectively called antagonism), and competition for nutrients and space. In addition, biocontrol has also been observed when Trichoderma is physically separated from the pathogen, thus implying an indirect systemic plant defence response. Therefore, in the second part, we describe the consecutive steps leading to induced systemic resistance (ISR), starting with the initial Trichoderma-plant interaction and followed by the activation of downstream signal transduction pathways and, ultimately, the defence response resulting in ISR (ISR-prime phase). Finally, we discuss the ISR-boost phase, representing the effect of ISR priming by Trichoderma spp. on plant responses after additional challenge with B. cinerea.

Influence of the Priming Effect with Alder Pollen on Nonspecific Bronchial Hyperreactivity of Asthmatics Specifically Allergic to Birch Pollen.

It has been shown that bronchial hyperreactivity in asthmatics specifically allergic to birch pollen is stable during the preseasonal and postseasonal periods and increases during the birch pollen season. Between January and March 1989, warm weather in the southern part of Sweden led to an early emission of hazel and alder pollens. Fourteen asthmatic patients living there were followed and demonstrated an increased nonspecific bronchial hyperreactivity (decreased PC20 methacholine) before the birch pollen season due to a 'priming effect' of related tree pollens.