Bacillus-based biocontrol beyond chemical control in central Africa: the challenge of turning myth into reality.

Agricultural productivity in the Great Lakes Countries of Central Africa, including Burundi, Rwanda, and the Democratic Republic of Congo, is affected by a wide range of diseases and pests which are mainly controlled by chemical pesticides. However, more than 30% of the pesticides used in the region are banned in European Union due to their high toxicity. Globally available safe and eco-friendly biological alternatives to chemicals are virtually non-existent in the region. Bacillus PGPR-based biocontrol products are the most dominant in the market and have proven their efficacy in controlling major plant diseases reported in the region. With this review, we present the current situation of disease and pest management and urge the need to utilize Bacillus-based control as a possible sustainable alternative to chemical pesticides. A repertoire of strains from the Bacillus subtilis group that have shown great potential to antagonize local pathogens is provided, and efforts to promote their use, as well as the search for indigenous and more adapted Bacillus strains to local agro-ecological conditions, should be undertaken to make sustainable agriculture a reality in the region.

Rhizomania: Hide and Seek of Polymyxa betae and the Beet Necrotic Yellow Vein Virus with Beta vulgaris.

The molecular interactions between Polymyxa betae, the protist vector of sugar beet viruses, beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania, and Beta vulgaris have not been extensively studied. Here, the transmission of BNYVV to sugar beet by P. betae zoospores was optimized using genetically characterized organisms. Molecular interactions of aviruliferous and viruliferous protist infection on sugar beet were highlighted by transcriptomic analysis. P. betae alone induced limited gene expression changes in sugar beet, as a biotrophic asymptomatic parasite. Most differentially expressed plant genes were down-regulated and included resistance gene analogs and cell wall peroxidases. Several enzymes involved in stress regulation, such as the glutathione-S-transferases, were significantly induced. With BNYVV, the first stages of the P. betae life cycle on sugar beet were accelerated with a faster increase of relative protist DNA level and an earlier appearance of sporangia and sporosori in plants roots. A clear activation of plant defenses and the modulation of genes involved in plant cell wall metabolism were observed. The P. betae transcriptome in the presence of BNYVV revealed induction of genes possibly involved in the switch to the survival stage. The interactions were different depending on the presence or absence of the virus. P. betae alone alleviates plant defense response, playing hide-and-seek with sugar beet and allowing for their mutual development. Conversely, BNYVV manipulates plant defense and promotes the rapid invasion of plant roots by P. betae. This accelerated colonization is accompanied by the development of thick-walled resting spores, supporting the virus survival. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Metagenomics approach for Polymyxa betae genome assembly enables comparative analysis towards deciphering the intracellular parasitic lifestyle of the plasmodiophorids.

Genomic knowledge of the tree of life is biased to specific groups of organisms. For example, only six full genomes are currently available in the rhizaria clade. Here, we have applied metagenomic techniques enabling the assembly of the genome of Polymyxa betae (Rhizaria, Plasmodiophorida) RES F41 isolate from unpurified zoospore holobiont and comparison with the A26-41 isolate. Furthermore, the first P. betae mitochondrial genome was assembled. The two P. betae nuclear genomes were highly similar, each with just ~10.2 k predicted protein coding genes, ~3% of which were unique to each isolate. Extending genomic comparisons revealed a greater overlap with Spongospora subterranea than with Plasmodiophora brassicae, including orthologs of the mammalian cation channel sperm-associated proteins, raising some intriguing questions about zoospore physiology. This work validates our metagenomics pipeline for eukaryote genome assembly from unpurified samples and enriches plasmodiophorid genomics; providing the first full annotation of the P. betae genome.

Spatio-temporal distribution of DMI and SDHI fungicide resistance of Zymoseptoria tritici throughout Europe based on frequencies of key target-site alterations.

BACKGROUND: Over the past decade, demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides have been extensively used to control to septoria tritici blotch, caused by Zymoseptoria tritici on wheat. This has led to the development and selection of alterations in the target-site enzymes (CYP51 and SDH, respectively). RESULTS: Taking advantage of newly and previously developed qPCR assays, the frequency of key alterations associated with DMI (CYP51-S524T) and SDHI (SDHC-T79N/I, C-N86S and C-H152R) resistance was assessed in Z. tritici-infected wheat leaf samples collected from commercial crops (n = 140) across 14 European countries prior to fungicide application in the spring of 2019. This revealed the presence of a West to East gradient in the frequencies of the most common key alterations conferring azole (S524T) and SDHI resistance (T79N and N86S), with the highest frequencies measured in Ireland and Great Britain. These observations were corroborated by sequencing (CYP51 and SDH subunits) and sensitivity phenotyping (prothioconazole-desthio and fluxapyroxad) of Z. tritici isolates collected from a selection of field samples. Additional sampling made at the end of the 2019 season confirmed the continued increase in frequency of the targeted alterations. Investigations on historical leaf DNA samples originating from different European countries revealed that the frequency of all key alterations (except C-T79I) has been gradually increasing over the past decade. CONCLUSION: Whilst these alterations are quickly becoming dominant in Ireland and Great Britain, scope still exists to delay their selection throughout the wider European population, emphasizing the need for the implementation of fungicide antiresistance measures. © 2021 Society of Chemical Industry.

Honey bee exposure scenarios to selected residues through contaminated beeswax.

Twenty-two pesticides and veterinary drugs of which residues were detected in beeswax in Europe were selected according to different criteria. The risk to honey bee health posed by the presence of these residues in wax was assessed based on three exposure scenarios. The first one corresponds to the exposure of larvae following their close contact with wax constituting the cells in which they develop. The second one corresponds to the exposure of larvae following consumption of the larval food that was contaminated from contact with contaminated wax. The third one corresponds to the exposure of adult honey bees following wax chewing when building cells and based on a theoretical worst-case scenario (= intake of contaminants from wax). Following these three scenarios, maximum concentrations which should not be exceeded in beeswax in order to protect honey bee health were calculated for each selected substance. Based on these values, provisional action limits were proposed. Beeswax exceeding these limits should not be put on the market.

Genomic and biological characterization of a novel partitivirus infecting Fusarium equiseti.

This study describes a new mycovirus infecting a strain from the Fusarium incarnatum-equiseti species complex. Based on phylogenetic and genomic analyses, this virus belongs to the recently proposed genus "Zetapartitivirus" in the family Partitiviridae. The name "Fusarium equiseti partitivirus 1″ (FePV1) is therefore suggested for this novel viral species. Similar to other partitiviruses, FePV1 genome is composed by two dsRNA segments that exhibit each one large ORF encoding for an RdRp and a CP, respectively. A smaller dsRNA was also detected in infected mycelium and could be a satellite RNA of FePV1. In addition to characterized zetapartitiviruses, other FePV1-related sequences were retrieved from online databases and their significance is discussed. Following conidial isolation, an FePV1-free isogenic line of the fungal host was obtained. In comparison with the original infected strain, this line showed higher growth, biomass production and pathogenicity on tomato, advocating that FePV1 induces hypovirulence on its host.

RNA silencing machinery contributes to inability of BSBV to establish infection in Nicotiana benthamiana: evidence from characterization of agroinfectious clones of Beet soil-borne virus.

Beet soil-borne virus (BSBV) is a sugar beet pomovirus frequently associated with Beet necrotic yellow veins virus, the causal agent of the rhizomania disease. BSBV has been detected in most of the major beet-growing regions worldwide, yet its impact on this crop remains unclear. With the aim to understand the life cycle of this virus and clarify its putative pathogenicity, agroinfectious clones have been engineered for each segment of its tripartite genome. The biological properties of these clones were then studied on different plant species. Local infection was obtained on agroinfiltrated leaves of Beta macrocarpa. On leaves of Nicotiana benthamiana, similar results were obtained, but only when heterologous viral suppressors of RNA silencing were co-expressed or in a transgenic line down regulated for both dicer-like protein 2 and 4. On sugar beet, local infection following agroinoculation was obtained on cotyledons, but not on other tested plant parts. Nevertheless, leaf symptoms were observed on this host via sap inoculation. Likewise, roots were efficiently mechanically infected, highlighting low frequency of root necrosis and constriction, and enabling the demonstration of transmission by the vector Polymyxa betae. Altogether, the entire viral cycle was reproduced, validating the constructed agroclones as efficient inoculation tools, paving the way for further studies on BSBV and its related pathosystem.

Description of a Novel Mycovirus in the Phytopathogen Fusarium culmorum and a Related EVE in the Yeast Lipomyces starkeyi.

A new mycovirus was found in the Fusarium culmorum strain A104-1 originally sampled on wheat in Belgium. This novel virus, for which the name Fusarium culmorum virus 1 (FcV1) is suggested, is phylogenetically related to members of the previously proposed family ''Unirnaviridae''. FcV1 has a monopartite dsRNA genome of 2898 bp that harbors two large non-overlapping ORFs. A typical -1 slippery motif is found at the end of ORF1, advocating that ORF2 is translated by programmed ribosomal frameshifting. While ORF2 exhibits a conserved replicase domain, ORF1 encodes for an undetermined protein. Interestingly, a hypothetically transcribed gene similar to unirnaviruses ORF1 was found in the genome of Lipomyces starkeyi, presumably resulting from a viral endogenization in this yeast. Conidial isolation and chemical treatment were unsuccessful to obtain a virus-free isogenic line of the fungal host, highlighting a high retention rate for FcV1 but hindering its biological characterization. In parallel, attempt to horizontally transfer FcV1 to another strain of F. culmorum by dual culture failed. Eventually, a screening of other strains of the same fungal species suggests the presence of FcV1 in two other strains from Europe.

Full genome sequence of a new polymycovirus infecting Fusarium redolens.

By screening a collection of Fusarium spp. for the presence of dsRNA, the Fusarium redolens strain A63-1 was found harboring a pattern of multiple dsRNA bands when analyzed by agarose gel electrophoresis. Using NextSeq Illumina sequencing, the full sequences of eight dsRNA molecules were determined, compared to databases, and gathered into a new viral genome. This novel virus shares similarities with mycoviruses that were recently grouped in the proposed family "Polymycoviridae". Hence, the name "Fusarium redolens polymycovirus 1" is proposed for this virus. Each viral dsRNA contains only one ORF, except dsRNA 7, which has an additional one. Based on amino acid sequence similarities, the functions of the proteins encoded by dsRNA 1-4 can be hypothesized. On the other hand, the putative proteins encoded by dsRNA 5-8 exhibit no relevant homology to known proteins. In this report, the full genome sequence of this new virus is presented along with a primary bioinformatics analysis.

First Draft Genome Sequence of a Polymyxa Genus Member, Polymyxa betae, the Protist Vector of Rhizomania.

Polymyxa betae belongs to the Plasmodiophorida (Phytomyxea, Rhizaria). Here, we report the first draft genome sequence of a member of the Polymyxa genus, which includes two obligate root endoparasite species, vectors of important soilborne plant viruses. The genome assembly was represented by 1,001 contigs, with a cumulated length of 27,085,946 bp.

The adaptation of Fusarium culmorum to DMI Fungicides Is Mediated by Major Transcriptome Modifications in Response to Azole Fungicide, Including the Overexpression of a PDR Transporter (FcABC1).

Fusarium culmorum is a fungal pathogen causing economically important diseases on a variety of crops. Fungicides can be applied to control this species with triazoles being the most efficient molecules. F. culmorum strains resistant to these molecules have been reported, but the underlying resistance mechanisms remain unknown. In this study, a tebuconazole-adapted F. culmorum strain was developed with a level of fitness similar to its parental strain. The adapted strain showed cross-resistance to all demethylation inhibitors (DMIs), but not to other classes of fungicides tested. RNA-Seq analysis revealed high transcriptomic differences between the resistant strain and its parental strain after tebuconazole treatment. Among these changes, FcABC1 (FCUL_06717), a pleiotropic drug resistance transporter, had a 30-fold higher expression level upon tebuconazole treatment in the adapted strains as compared to the wild-type strain. The implication of this transporter in triazole resistance was subsequently confirmed in field strains harboring distinct levels of sensitivity to triazoles. FcABC1 is present in other species/genera, including F. graminearum in which it is known to be necessary for azole resistance. No difference in FcABC1 sequences, including the surrounding regions, were found when comparing the resistant strain to the wild-type strain. Fusarium culmorum is therefore capable to adapt to triazole pressure by overexpressing a drug resistance transporter when submitted to triazoles and the same mechanism is anticipated to occur in other species.

Long Term Management of Rhizomania Disease-Insight Into the Changes of the Beet necrotic yellow vein virus RNA-3 Observed Under Resistant and Non-resistant Sugar Beet Fields.

Rhizomania disease, caused by the Beet necrotic yellow vein virus (BNYVV), is considered as one of the major constraints for sugar beet production, worldwide. As a result of the introgression of major resistance genes (Holly, Rz2) in commercially available sugar beet varieties, the virus has endured strong selection pressure since the 90s'. Understanding the virus response and diversity to sugar beet resistance is a key factor for a sustainable management of only few resistance genes. Here we report rhizomania surveys conducted in a rhizomania hot spot, the Pithiviers area (France) during a 4-year period and complementary to the study of Schirmer et al. (2005). The study aimed at evaluating the intra- and inter-field BNYVV diversity in response to different sources of resistance and over the growing season. To follow rhizomania development over the sugar beet growing season, extensive field samplings combined with field assays were performed in this study. The evolution of the BNYVV diversity was assessed at intra- and inter-field levels, with sugar beet cultivars containing different resistance genes (Rz1, Rz1 + Heterodera schachtii resistance and Rz1Rz2). Intra-field diversity was analyzed at the beginning and the end of the growing season of each field. From more than one thousand field samples, the simultaneous presence of the different A, B and P types of BNYVV was confirmed, with 21 variants identified at positions 67-70 of the p25 tetrad. The first variant, AYHR, was found most commonly followed by SYHG. Numerous mixed infections (9.93% of the samples), mostly of B-type with P-type, have also been evidenced. Different tetrads associated with the A- or B-type were also found with a fifth RNA-genome component known to allow more aggressiveness to BNYVV on sugar beet roots. Cultivars with Rz1+Rz2 resistant genes showed few root symptoms even if the BNYVV titre was quite high according to the BNYVV type present. The virus infectious potential in the soil at the end of the growing season with such cultivars was also lower despite a wider diversity at the BNYVV RNA3 sequence level. Rz1+Rz2 cultivars also exhibited a lower presence of Beet soil-borne virus (BSBV), a P. betae-transmitted Pomovirus. Cultivars with Rz1 and nematode (N) resistance genes cultivated in field infected with nematodes showed lower BNYVV titre than those with Rz1 or Rz1+Rz2 cultivars. Overall, the population structure of BNYVV in France is shown to be different from that previously evidenced in different world areas. Implications for long-term management of the resistance to rhizomania is discussed.

Versatile Antagonistic Activities of Soil-Borne Bacillus spp. and Pseudomonas spp. against Phytophthora infestans and Other Potato Pathogens.

The world potato is facing major economic losses due to disease pressure and environmental concerns regarding pesticides use. This work aims at addressing these two issues by isolating indigenous bacteria that can be integrated into pest management strategies. More than 2,800 strains of Bacillus-like and Pseudomonas-like were isolated from several soils and substrates associated with potato agro-systems in Belgium. Screenings for antagonistic activities against the potato pathogens Alternaria solani, Fusarium solani (BCCM-MUCL 5492), Pectobacterium carotovorum (ATCC 15713), Phytophthora infestans (CRA-W10022) and Rhizoctonia solani (BCCM-MUCL 51929) were performed, allowing the selection of 52 Bacillus spp. and eight Pseudomonas spp. displaying growth inhibition of at least 50% under in vitro conditions, particularly against P. infestans. All 60 bacterial isolates were identified based on 16S rRNA gene sequencing and further characterized for the production of potential bio-active secondary metabolites. The antagonistic activities displayed by the selected strains indicated that versatile metabolites can be produced by the strains. For instance, the detection of genes involved bacilysin biosynthesis was correlated with the strong antagonism of Bacillus pumilus strains toward P. infestans, whereas the production of both bio-surfactants and siderophores might explain the high antagonistic activities against late blight. Greenhouse assays with potato plants were performed with the most effective strains (seven Bacillus spp. and four Pseudomonas spp.) in order to evaluate their in vivo antagonistic effect against P. infestans. Based on these results, four strains (Bacillus amyloliquefaciens 17A-B3, Bacillus subtilis 30B-B6, Pseudomonas brenneri 43R-P1 and Pseudomonas protegens 44R-P8) were retained for further evaluation of their protection index against P. infestans in a pilot field trial. Interestingly, B. subtilis 30B-B6 was shown to significantly decrease late blight severity throughout the crop season. Overall, this study showed that antagonistic indigenous soil bacteria can offer an alternative to the indiscriminate use of pesticide in potato agro-systems.

Assessment of Tomato (Solanum lycopersicum L.) Producers' Exposure Level to Pesticides, in Kouka and Toussiana (Burkina Faso).

To assess producers' exposure level to pesticides in vegetable production in Burkina Faso, a study was carried out in 2016 and 2017 among 30 tomato producers in the municipalities of Kouka and Toussiana. Eighteen (18) commercial formulations were identified, with more than 50% of pesticides destined for cotton production. Eleven active substances have been identified and the most frequently used are λ-cyhalothrin (35%), acetamiprid (22%) and profenofos (13%). The most commonly used chemical families are pyrethroids (28%) and organophosphates (18%). The study revealed a low level of training for producers, a high use of pesticides according to the Frequency Treatment Indicator, and a very low level of protection used by producers. The Health Risk Index shows that active substances such as methomyl, λ-cyhalothrin and profenofos present very high risk to operators' health. Based on the UK-POEM model, the predictive exposure levels obtained varied from 0.0105 mg/kg body weight/day to 1.7855 mg/kg body weight/day, which is several times higher than the Acceptable Operator Exposure Level. However, the study also shows that exposure can be greatly reduced if the required Personal Protective Equipment is worn. Producers' awareness and training on integrated pest management are necessary to reduce the risks linked to the pesticides use in Burkina Faso.

Not in your usual Top 10: protists that infect plants and algae.

Fungi, nematodes and oomycetes belong to the most prominent eukaryotic plant pathogenic organisms. Unicellular organisms from other eukaryotic lineages, commonly addressed as protists, also infect plants. This review provides an introduction to plant pathogenic protists, including algae infecting oomycetes, and their current state of research.

Sensitivity of Fusarium culmorum to triazoles: impact of trichothecene chemotypes, oxidative stress response and genetic diversity.

BACKGROUND: Fusarium culmorum is a fungal pathogen occurring worldwide on various weeds and important crops. Triazoles have been shown to be the most effective fungicide for managing Fusarium spp., but little is known about their specific activity on F. culmorum. RESULTS: The sensitivity of 107 F. culmorum strains to triazoles was assessed using microtitre plate assays. The EC50 values ranged from 0.14 to 1.53 mg L-1 for tebuconazole and from 0.25 to 2.47 mg L-1 for epoxiconazole. Cross-resistance to both azoles was found (r = 0.61). F. culmorum appeared to be significantly more sensitive than F. graminearum or F. cerealis. No increase in the mean EC50 was observed over time, which might be related to an unfavourable fitness cost, measured here as fungal growth. On average, nivalenol-producing strains of F. culmorum were significantly more resistant than deoxynivalenol-producing strains. The relationship between resistance and chemotype-dependent adaptation to oxidative stress was investigated, but remained unclear. No link between inter-simple sequence repeat (ISSR) genetic diversity and triazole resistance could be established. CONCLUSION: Fungicide use might not be a driving force in the evolution of F. culmorum, and the benefit of a resistance trait probably does not outweigh its costs. © 2016 Society of Chemical Industry.

Residues in Beeswax: A Health Risk for the Consumer of Honey and Beeswax?

A scenario analysis in regard to the risk of chronic exposure of consumers to residues through the consumption of contaminated honey and beeswax was conducted. Twenty-two plant protection products and veterinary substances of which residues have already been detected in beeswax in Europe were selected. The potential chronic exposure was assessed by applying a worst-case scenario based on the addition of a "maximum" daily intake through the consumption of honey and beeswax to the theoretical maximum daily intake through other foodstuffs. For each residue, the total exposure was finally compared to the acceptable daily intake. It is concluded that the food consumption of honey and beeswax contaminated with these residues considered separately does not compromise the consumer's health, provided proposed action limits are met. In regard to residues of flumethrin in honey and in beeswax, "zero tolerance" should be applied.

Relationship between Fusarium spp. diversity and mycotoxin contents of mature grains in southern Belgium.

Over a 4-year period (2010-13), a survey aiming at determining the occurrence of Fusarium spp. and their relations to mycotoxins in mature grains took place in southern Belgium. The most prevalent species were F. graminearum, F. avenaceum, F. poae and F. culmorum, with large variations between years and locations. An even proportion of mating type found for F. avenaceum, F. culmorum, F. cerealis and F. tricinctum is usually a sign of ongoing sexual recombination. In contrast, an unbalanced proportion of mating type was found for F. poae and no MAT1-2 allele was present in the F. langsethiae population. Genetic chemotyping indicates a majority of deoxynivalenol (DON)-producing strains in F. culmorum (78%, all 3-ADON producers) and F. graminearum (95%, mostly 15-ADON producers), while all F. cerealis strains belong to the nivalenol (NIV) chemotype. Between 2011 and 2013, DON, NIV, enniatins (ENNs) and moniliformin (MON) were found in each field in various concentrations. By comparison, beauvericin (BEA) was scarcely detected and T-2 toxin, zearalenone and α- and ß-zearalenols were never detected. Principal component analysis revealed correlations of DON with F. graminearum, ENNs and MON with F. avenaceum and NIV with F. culmorum, F. cerealis and F. poae. BEA was associated with the presence of F. tricinctum and, to a lesser extent, with the presence of F. poae. The use of genetic chemotype data revealed that DON concentrations were mostly influenced by DON-producing strains of F. graminearum and F. culmorum, whereas the concentrations of NIV were influenced by the number of NIV-producing strains of both species added to the number of F. cerealis and F. poae strains. This study emphasises the need to pay attention to less-studied Fusarium spp. for future Fusarium head blight management strategies, as they commonly co-occur in the field and are associated with a broad spectrum of mycotoxins.

Systemic resistance induced by Bacillus lipopeptides in Beta vulgaris reduces infection by the rhizomania disease vector Polymyxa betae.

The control of rhizomania, one of the most important diseases of sugar beet caused by the Beet necrotic yellow vein virus, remains limited to varietal resistance. In this study, we investigated the putative action of Bacillus amylolequifaciens lipopeptides in achieving rhizomania biocontrol through the control of the virus vector Polymyxa betae. Some lipopeptides that are produced by bacteria, especially by plant growth-promoting rhizobacteria, have been found to induce systemic resistance in plants. We tested the impact of the elicitation of systemic resistance in sugar beet through lipopeptides on infection by P. betae. Lipopeptides were shown to effectively induce systemic resistance in both the roots and leaves of sugar beet, resulting in a significant reduction in P. betae infection. This article provides the first evidence that induced systemic resistance can reduce infection of sugar beet by P. betae.

Acquisition and transmission of Peanut clump virus by Polymyxa graminis on cereal species.

The objective of this study was to investigate the specificity of the interactions between Polymyxa graminis, Peanut clump virus (PCV), and cereals, particularly the acquisition and the transmission of the virus by three P. graminis formae speciales. A new strategy has been developed: it involves using sugarcane as the common host for both the virus and its vector in order to produce the viruliferous zoospores of P. graminis f. sp. subtropicalis, temperata, and tropicalis that were then inoculated on cereal species. This experiment enabled the role of P. graminis f. sp. tropicalis and subtropicalis zoospores in PCV transmission to be demonstrated. The efficiency of this transmission was shown to vary, depending on the P. graminis special forms. Interestingly, the high transmission of the PCV isolate from Burkina Faso by an isolate of P. graminis f. sp. tropicalis from Niger on pearl millet suggests that there is a coevolution mechanism in this pathosystem. The study also provides evidence that the host plant species in which Polymyxa zoospores are produced could affect the infectivity of the vector. Finally, using Polymyxa quantitation by quantitative reverse-transcription polymerase chain reaction and in situ observations of the virus, the study demonstrates the independence of the development of PCV and its vector in the host plants.