Meeting Report of the Second Symposium of the Belgian Society for Viruses of Microbes and Launch of the Phage Valley.

The second symposium of the Belgian Society for Viruses of Microbes (BSVoM) took place on 8 September 2023 at the University of Liège with 141 participants from 10 countries. The meeting program covered three thematic sessions opened by international keynote speakers: two sessions were devoted to "Fundamental research in phage ecology and biology" and the third one to the "Present and future applications of phages". During this one day symposium, four invited keynote lectures, nine selected talks and eight student pitches were given along with thirty presented posters. The president of the Belgian Society for Viruses of Microbes, Prof. Yves Briers, took advantage of this symposium to launch the Phage Valley concept that will put the spotlight on the exceptionally high density of researchers investigating viruses of microbes as well as the successful triple helix approach between academia, industry and government in Belgium.

Foundation of the Belgian Society for Viruses of Microbes and Meeting Report of Its Inaugural Symposium.

The Belgian Society for Viruses of Microbes (BSVoM) was founded on 9 June 2022 to capture and enhance the collaborative spirit among the expanding community of microbial virus researchers in Belgium. The sixteen founders are affiliated to fourteen different research entities across academia, industry and government. Its inaugural symposium was held on 23 September 2022 in the Thermotechnical Institute at KU Leuven. The meeting program covered three thematic sessions launched by international keynote speakers: (1) virus-host interactions, (2) viral ecology, evolution and diversity and (3) present and future applications. During the one-day symposium, four invited keynote lectures, ten selected talks and eight student pitches were given along with 41 presented posters. The meeting hosted 155 participants from twelve countries.

First report of black rot disease in Eruca vesicaria subsp. sativa caused by Xanthomonas campestris pv. campestris in Belgium.

Eruca vesicaria subsp. sativa (Mill.) Thell. (arugula or rocket) is a leafy vegetable originating from the Mediterranean region primarily being sold in bagged salads. From 2014 to 2017, plants (cv. Montana) exhibiting blackened leaf veins and irregular V-shaped chlorotic to necroic lesions at the leaf margins were observed in commercial greenhouses in Flanders, Belgium (Figure S1A). Symptoms started after harvest of the first cut, indicating that leaf injury favours disease development. By the last cut, infections had spread uniformly across the plots, with symptoms advanced to the point where harvesting was no longer profitable. Excised surface-sterilized necrotic leaf tissue and seeds were homogenized in phosphate buffer (PB), followed by dilution plating on Pseudomonas Agar F containing sucrose. After four days at 28°C, bright yellow round, mucoid, convex Xanthomonas-like colonies were obtained, both from leaves and seeds. For confirmation, DNA was extracted from pure cultures after which a partial fragment of gyrB was amplified and sequenced (Holtappels et al. 2022). Amplicons were trimmed to 530 nucleotides (Genbank ON815895-ON815900) according to Parkinson et al. (2007) and compared with the NCBI database. Strain GBBC 3139 shares 100% sequence identity with Xanthomonas campestris pv. campestris (Xcc) type strain LMG 568 and with RKFB 1361-1364, isolated from arugula in Serbia (Prokic et al. 2022). The other isolates from Belgian rocket - GBBC 3036, 3058, 3077, 3217 and 3236 - all have a gyrB sequence 100% identical to that of Xcc strain ICMP 4013, among others. To determine the genetic relatedness to other pathogenic Xc strains, the genomes of GBBC 3077, 3217, 3236 and 3139 were sequenced using a MinION (Nanopore) and non-clonal sequences were submitted to NCBI (BioProject PRJNA967242). Genomes were compared by calculating Average Nucleotide Identity (ANI). This revealed that the Belgian strains cluster together with Xc isolates originating from Brassica crops and separate from strains identified as Xc pv. barbareae, pv. incanae and pv. raphani (Figure S2A). Their designation as pv. campestris is supported by maximum likelihood clustering of concatenated gyrB-avrBs2 sequences (EPPO, 2021; Figure S2B,C). Finally, pathogenicity was verified on five-week-old rocket 'Pronto' plants grown in a commercial potting mix by cutting the leaves along the midrib with scissors dipped into a suspension of 108 cfu/ml of each strain or PB as control (4 plants/strain). Plants were kept in closed polypropylene boxes for 48 hr to support high humidity and facilitate infection. They were then maintained at 25 ± 2 °C. Lesions like those observed on commercial plants developed on the inoculated leaves within one week (Figure S1B). Bacterial colonies reisolated from symptomatic tissue were identified based on gyrB as the strains used for inoculation, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of black rot disease in arugula caused by Xcc in Belgium. Previously, Xcc on arugula has been reported in Argentina, California and Serbia as well (Romero et al. 2008; Rosenthal et al. 2017; Prokic et al. 2022). Arugula being a minor crop in Belgium, challenged by Xcc infections and strong import competition, many growers have abandoned the sector in recent years. Therefore, this study makes a strong case for early detection of disease symptoms and timely application of relevant management strategies in vulnerable crop settings.

Respiratory CO2 Combined With a Blend of Volatiles Emitted by Endophytic Serendipita Strains Strongly Stimulate Growth of Arabidopsis Implicating Auxin and Cytokinin Signaling.

Rhizospheric microorganisms can alter plant physiology and morphology in many different ways including through the emission of volatile organic compounds (VOCs). Here we demonstrate that VOCs from beneficial root endophytic Serendipita spp. are able to improve the performance of in vitro grown Arabidopsis seedlings, with an up to 9.3-fold increase in plant biomass. Additional changes in VOC-exposed plants comprised petiole elongation, epidermal cell and leaf area expansion, extension of the lateral root system, enhanced maximum quantum efficiency of photosystem II (Fv/Fm), and accumulation of high levels of anthocyanin. Notwithstanding that the magnitude of the effects was highly dependent on the test system and cultivation medium, the volatile blends of each of the examined strains, including the references S. indica and S. williamsii, exhibited comparable plant growth-promoting activities. By combining different approaches, we provide strong evidence that not only fungal respiratory CO2 accumulating in the headspace, but also other volatile compounds contribute to the observed plant responses. Volatile profiling identified methyl benzoate as the most abundant fungal VOC, released especially by Serendipita cultures that elicit plant growth promotion. However, under our experimental conditions, application of methyl benzoate as a sole volatile did not affect plant performance, suggesting that other compounds are involved or that the mixture of VOCs, rather than single molecules, accounts for the strong plant responses. Using Arabidopsis mutant and reporter lines in some of the major plant hormone signal transduction pathways further revealed the involvement of auxin and cytokinin signaling in Serendipita VOC-induced plant growth modulation. Although we are still far from translating the current knowledge into the implementation of Serendipita VOCs as biofertilizers and phytostimulants, volatile production is a novel mechanism by which sebacinoid fungi can trigger and control biological processes in plants, which might offer opportunities to address agricultural and environmental problems in the future.

Evaluation of genome size and quantitative features of the dolipore septum as taxonomic predictors for the Serendipita 'williamsii' species complex.

Despite multiple taxonomic revisions, several uncertainties at the genus and species level remain to be resolved within the Serendipitaceae family (Sebacinales). This volatile classification is attributed to the limited number of available axenic cultures and the scarcity of useful morphological traits. In the current study, we attempted to discover alternative taxonomic markers not relying on DNA sequences to differentiate among the closely related members of our Congolese Serendipita isolate collection and the reference strains S. indica (syn. Piriformospora indica) and S. williamsii (syn. P. williamsii). We demonstrated that nuclear distribution across hyphal cells and genome size (determined by flow cytometry) did not have enough resolving power, but quantitative and qualitative variations in the ultrastructure of the dolipore septa investigated by transmission electron microscopy did provide useful markers. Multivariate analysis revealed that subtle differences in ultrastructural characteristics of the parenthesome and the attached endoplasmic reticulum are most relevant when studying this fungal group. Moreover, the observed clustering pattern showed that there might be more diversity amongst the Congolese isolates within the S. 'williamsii' species complex than previously anticipated based on molecular data. Altogether, our results provide novel perspectives on the use of integrative approaches to support sebacinoid and Serendipitaceae taxonomy.

Functional Genomics Insights Into the Pathogenicity, Habitat Fitness, and Mechanisms Modifying Plant Development of Rhodococcus sp. PBTS1 and PBTS2.

Pistachio Bushy Top Syndrome (PBTS) is a recently emerged disease that has strongly impacted the pistachio industry in California, Arizona, and New Mexico. The disease is caused by two bacteria, designated PBTS1 that is related to Rhodococcus corynebacterioides and PBTS2 that belongs to the species R. fascians. Here, we assessed the pathogenic character of the causative agents and examined their chromosomal sequences to predict the presence of particular functions that might contribute to the observed co-occurrence and their effect on plant hosts. In diverse assays, we confirmed the pathogenicity of the strains on "UCB-1" pistachio rootstock and showed that they can also impact the development of tobacco species, but concurrently inconsistencies in the ability to induce symptoms were revealed. We additionally evidence that fas genes are present only in a subpopulation of pure PBTS1 and PBTS2 cultures after growth on synthetic media, that these genes are easily lost upon cultivation in rich media, and that they are enriched for in an in planta environment. Analysis of the chromosomal sequences indicated that PBTS1 and PBTS2 might have complementary activities that would support niche partitioning. Growth experiments showed that the nutrient utilization pattern of both PBTS bacteria was not identical, thus avoiding co-inhabitant competition. PBTS2 appeared to have the potential to positively affect the habitat fitness of PBTS1 by improving its resistance against increased concentrations of copper and penicillins. Finally, mining the chromosomes of PBTS1 and PBTS2 suggested that the bacteria could produce cytokinins, auxins, and plant growth-stimulating volatiles and that PBTS2 might interfere with ethylene levels, in support of their impact on plant development. Subsequent experimentation supported these in silico predictions. Altogether, our data provide an explanation for the observed pathogenic behavior and unveil part of the strategies used by PBTS1 and PBTS2 to interact with plants.

Sebacinoids within rhizospheric fungal communities associated with subsistence farming in the Congo Basin: a needle in each haystack.

The unique ecosystem of the Congolese rainforest has only scarcely been explored for its plant-fungal interactions. Here, we characterized the root fungal communities of field-grown maize and of Panicum from adjacent borders in the Congo Basin and assessed parameters that could shape them. The soil properties indicated that comparable poor soil conditions prevailed in fields and borders, illustrating the low input character of local subsistence farming. The rhizosphere fungal communities, dominated by ascomycetous members, were structured by plant species, slash-and-burn practices and soil P, pH and C/N ratio. Examining fungi with potential plant growth-promoting abilities, the glomeromycotan communities appeared to be affected by the same parameters, whereas the inconspicuous symbionts of the order Sebacinales seemed less susceptible to environmental and anthropogenic factors. Notwithstanding the low abundances at which they were detected, sebacinoids occurred in 87% of the field samples, implying that they represent a consistent taxon within indigenous fungal populations across smallholder farm sites. Pending further insight into their ecosystem functionality, these data suggest that Sebacinales are robust root inhabitants that might be relevant for on-farm inoculum development within sustainable soil fertility management in the Sub-Saharan region.

Congolese Rhizospheric Soils as a Rich Source of New Plant Growth-Promoting Endophytic Piriformospora Isolates.

In the last decade, there has been an increasing focus on the implementation of plant growth-promoting (PGP) organisms as a sustainable option to compensate for poor soil fertility conditions in developing countries. Trap systems were used in an effort to isolate PGP fungi from rhizospheric soil samples collected in the region around Kisangani in the Democratic Republic of Congo. With sudangrass as a host, a highly conducive environment was created for sebacinalean chlamydospore formation inside the plant roots resulting in a collection of 51 axenically cultured isolates of the elusive genus Piriformospora (recently transferred to the genus Serendipita). Based on morphological data, ISSR fingerprinting profiles and marker gene sequences, we propose that these isolates together with Piriformospora williamsii constitute a species complex designated Piriformospora (= Serendipita) 'williamsii.' A selection of isolates strongly promoted plant growth of in vitro inoculated Arabidopsis seedlings, which was evidenced by an increase in shoot fresh weight and a strong stimulation of lateral root formation. This isolate collection provides unprecedented opportunities for fundamental as well as translational research on the Serendipitaceae, a family of fungal endophytes in full expansion.