Feeding selectivity of Aphelenchoides besseyi and A. pseudogoodeyi on fungi associated with Florida strawberry

Aphelenchoides besseyi and A. pseudogoodeyi are foliar nematodes associated with commercial strawberry production in Florida, USA. The reproductive and feeding habits of these two nematode species were assessed on Florida isolates of the fungi Botrytis cinerea, Colletotrichum gloeosporioides, Macrophomina phaseolina, and Neopestalotiopsis spp. pathogenic to strawberry, and the non-pathogenic isolates of Fusarium oxysporum and Monilinia fructicola grown on PDA in petri dishes. Each culture was inoculated with six specimens with mix life stages of either A. besseyi and A. pseudogoodeyi and incubated at 24°C under axenic and non-axenic conditions 23 and 31 days after inoculation, respectively. Aphelenchoides besseyi reproduction rates were significantly higher on strawberry pathogenic isolates of B. cinerea, C. gloeosporioides, and Neopestalotiopsis rosae than on the non-pathogenic isolates of F. oxysporum and M. fructicola. In contrast, reproductive rates of A. pseudogoodeyi did not significantly vary across the fungi cultures. For both nematode species, Macrophomina phaseolina was a poor host because it did not produce mycelium on the media used. Our findings indicate that A. besseyi is more selective in its fungal-feeding preference than A. pseudogoodeyi. Additionally, A. pseudogoodeyi eggs and juveniles were significantly higher than adults. Yet, for A. besseyi, adult stages were more common. Fungi aid in the maintenance of soil-dwelling populations of these two nematode species. Removing fungi-infected strawberry plant residues is both a desirable and effective management practice to limit A. besseyi in central Florida commercial strawberry fields.

UPLC-QTOF-MS-Based Untargeted Study of Metabolites Secreted by Sclerotinia Sclerotiorum Pathogen Under Axenic Condition

The stem rot disease has emerged globally as a major disease threat to the productivity and seed quality of oilseed Brassica. The generalist causal pathogen Sclerotinia sclerotiorum (Lib.) de Bary shows large variability in their aggressiveness and pathogenicity. Revealing the metabolic profile and signaling components of the pathogen in host-pathogen interaction are fundamental in understanding host resistance to the disease. In this study, the metabolites released by the pathogenic strains of S. sclerotiorum under the axenic culture have been identified using the untargeted high-resolution UPLC-QTOF-ESI-MS/MS. The analysis of the ethyl acetate extracts of the S. sclerotiorum culture revealed ten major secondary metabolites namely, sclerin, sclerotinin-B, sclerone, melanin, bostrycoidin, botcinin-D, botcinin-A, gliovirin, scleramide, and botcinic acid. The later six metabolites are being reported for the first time in the culture extract of the S. sclerotiorum pathogen. The phylogenetic analysis based on the overlapping and unique informative peaks in the chromatograms, the six S. sclerotiorum strains were grouped into three major clads. The clustering based on metabolic profiles does not substantiate the diversity based on morphology or virulence differences on the host. The findings of the study signified the metabolites secreted under the axenic conditions are varies based on their growth and developmental stages and may not necessarily be the determining factors for their differential aggressiveness and virulence over the host.

Haemocytes are critical for Drosophila melanogaster post-embryonic development, independent of control of the microbiota

Proven roles for haemocytes (blood cells) have expanded beyond the control of infections in Drosophila. Despite this, the critical role of haemocytes in post-embryonic development has long been thought to be limited to control of microorganisms during metamorphosis. This has previously been shown by rescue of adult development in haemocyte-ablation models under germ-free conditions. Here we show that haemocytes have a critical role in post-embryonic development beyond their ability to control the microbiota. Using a newly generated, strong haemocyte-specific driver line for the GAL4/UAS system, we show that specific ablation of haemocytes is pupal lethal, even under axenic conditions. Genetic rescue experiments prove that this is a haemocyte-specific phenomena. RNA-seq data suggests that dysregulation of the midgut is a critical consequence of haemocyte ablation. We believe this novel role of haemocytes during metamorphosis is a major finding for the field. This is an exciting new Drosophila model to study the precise mechanisms in which haemocytes regulate tissue development, findings from which could have far reaching implications beyond invertebrate biology.

Drice restrains Diap2-mediated inflammatory signalling and intestinal inflammation

The Drosophila IAP protein, Diap2, is a key mediator of NF-κB signalling and innate immune responses. Diap2 is required for both local immune activation, taking place in the epithelial cells of the gut and trachea, and for mounting systemic immune responses in the cells of the fat body. We have found that transgenic expression of Diap2 leads to a spontaneous induction of NF-κB target genes, inducing chronic inflammation in the Drosophila midgut, but not in the fat body. Drice is a Drosophila effector caspase known to interact and form a stable complex with Diap2. We have found that this complex formation induces its subsequent degradation, thereby regulating the amount of Diap2 driving NF-κB signalling in the intestine. Concordantly, loss of Drice activity leads to accumulation of Diap2 and to chronic intestinal inflammation. Interestingly, Drice does not interfere with pathogen-induced signalling, suggesting that it protects from immune responses induced by resident microbes. Accordingly, no inflammation was detected in transgenic Diap2 flies and Drice-mutant flies reared in axenic conditions. Hence, we show that Drice, by restraining Diap2, halts unwanted inflammatory signalling in the intestine.

Mycobacterium tuberculosis Toxin CpnT Is an ESX-5 Substrate and Requires Three Type VII Secretion Systems for Intracellular Secretion

ABSTRACT CpnT, a NAD+ glycohydrolase, is the only known toxin that is secreted by Mycobacterium tuberculosis. CpnT is composed of two domains; the C-terminal domain is the toxin, whereas the N-terminal domain is required for secretion. CpnT shows characteristics of type VII secretion (T7S) substrates, including a predicted helix-turn-helix domain followed by a secretion motif (YxxxE). Disruption of this motif indeed abolished CpnT secretion. By analyzing different mutants, we established that CpnT is specifically secreted by the ESX-5 system in Mycobacterium marinum under axenic conditions and during macrophage infection. Surprisingly, intracellular secretion of CpnT was also dependent on both ESX-1 and ESX-4. These secretion defects could be partially rescued by coinfection with wild-type bacteria, indicating that secreted effectors are involved in this process. In summary, our data reveal that three different type VII secretion systems have to be functional in order to observe intracellular secretion of the toxin CpnT. IMPORTANCE For decades, it was believed that the intracellular pathogen M. tuberculosis does not possess toxins. Only fairly recently it was discovered that CpnT is a potent secreted toxin of M. tuberculosis, causing necrotic cell death in host cells. However, until now the secretion pathway remained unknown. In our study, we were able to identify CpnT as a substrate of the mycobacterial type VII secretion system. Pathogenic mycobacteria have up to five different type VII secretion systems, called ESX-1 to ESX-5, which play distinct roles for the pathogen during growth or infection. We were able to elucidate that CpnT is exclusively secreted by the ESX-5 system in bacterial culture. However, to our surprise we discovered that, during infection studies, CpnT secretion relies on intact ESX-1, ESX-4, and ESX-5 systems. We elucidate for the first time the intertwined interplay of three different and independent secretion systems to secrete one substrate during infection.

Bacteria enhance the production of extracellular polymeric substances by the green dinoflagellate Lepidodinium chlorophorum

High biomasses of the marine dinoflagellate Lepidodinium chlorophorum cause green seawater discolorations along Southern Brittany (NE Atlantic, France). The viscosity associated to these phenomena has been related to problems in oyster cultivation. The harmful effect of L. chlorophorum might originate from the secretion of Extracellular Polymeric Substances (EPS). To understand whether the EPS are produced by L. chlorophorum or its associated bacteria, or if they are a product of their interaction, batch cultures were performed under non-axenic and pseudo-axenic conditions for three strains. Maximum dinoflagellate cell abundances were observed in pseudo-axenic cultures. The non-sinking fraction of polymers (Soluble Extracellular Polymers, SEP), mainly composed of proteins and the exopolysaccharide sulphated galactan, slightly increased in pseudo-axenic cultures. The amount of Transparent Exopolymer Particles (TEP) per cell increased under non-axenic conditions. Despite the high concentrations of Particulate Organic Carbon (POC) measured, viscosity did not vary. These results suggest that the L. chlorophorum-bacteria interaction could have a detrimental consequence on the dinoflagellate, translating in a negative effect on L. chlorophorum growth, as well as EPS overproduction by the dinoflagellate, at concentrations that should not affect seawater viscosity.

Bio-Guided Isolation of Antimalarial Metabolites from the Coculture of Two Red Sea Sponge-Derived Actinokineospora and Rhodococcus spp.

Coculture is a productive technique to trigger microbes’ biosynthetic capacity by mimicking the natural habitats’ features principally by competition for food and space and interspecies cross-talks. Mixed cultivation of two Red Sea-derived actinobacteria, Actinokineospora spheciospongiae strain EG49 and Rhodococcus sp. UR59, resulted in the induction of several non-traced metabolites in their axenic cultures, which were detected using LC–HRMS metabolomics analysis. Antimalarial guided isolation of the cocultured fermentation led to the isolation of the angucyclines actinosporins E (1), H (2), G (3), tetragulol (5) and the anthraquinone capillasterquinone B (6), which were not reported under axenic conditions. Interestingly, actinosporins were previously induced when the axenic culture of the Actinokineospora spheciospongiae strain EG49 was treated with signalling molecule N-acetyl-d-glucosamine (GluNAc); this finding confirmed the effectiveness of coculture in the discovery of microbial metabolites yet to be discovered in the axenic fermentation with the potential that could be comparable to adding chemical signalling molecules in the fermentation flask. The isolated angucycline and anthraquinone compounds exhibited in vitro antimalarial activity and good biding affinity against lysyl-tRNA synthetase (PfKRS1), highlighting their potential developability as new antimalarial structural motif.

Updated Characterization of Races of Plasmopara halstedii and Entomopathogenic Fungi as Endophytes of Sunflower Plants in Axenic Culture

The management of downy mildew (Plasmopara halstedii) in sunflower, is heavily dependent on genetic resistance, whilst entomopathogenic fungi (EF) can reduce other sunflower diseases. In this work, we characterized P. halstedii from Spain and other countries collected in the past few years. Twenty-three races were identified (the most frequent in Spain being 310, 304, 705 and 715), with an increasing proportion of highly virulent races. Five isolates from countries other than Spain overcame the resistance in RHA-340. In addition, we assessed the efficacy of five EF against downy mildew and their effects on sunflower growth in axenic conditions. None of the entomopathogens reduced disease severity, nor did they have any effect on plant growth when applied together with P. halstedii. In contrast, three EF reduced some of the plant growth variables in the absence of the pathogen. Microbiological and molecular diagnostics suggest that the axenic system and the short experimental time used in this study did not favor the successful establishment of EF in the plants or their potential biocontrol effect. Our results show a shift in P. halstedii racial patterns and suggest that soil as a growth substrate and long infection times are needed for EF effectiveness against downy mildew.