War in the darkness: Association of semiochemicals and entomopathogens to control wireworms

Wireworms (Coleoptera: Elateridae) are generalist insect pests attacking a wide range of plants, causing significant damages in agriculture. They feed on seeds, roots or crown and are particularly impactful on young seedlings. Wireworms locate their hosts using different cues including Volatiles Organic Compounds (VOCs) released in the rhizosphere and guiding the larvae to an appropriate host. In this study, we aimed at developing an Attract-and-kill strategy by setting up two distinct objectives : (1) comparing the attraction potential of different semiochemicals and (2) identify strains of entomopathogenic fungi and nematodes that are efficient in killing wireworms. Plant natural extracts were encapsulated in alginate beads and were shown to attract up to 76% of the tested wireworms, during behavioral assays performed in two-way olfactometers. We then performed volatile collection on these alginate beads and revealed that beads released a blend of eight VOCs, of which acetoin was the one released in higher quantity. Finally, the virulence of twelve strains of entomopathogenic fungi and eleven strains of entomopathogenic nematodes was evaluated. Two species of Metarhizium and one species of Ophiocordyceps were the most virulent against wireworms, leading to mortality levels of 39%, 50% and 57%, respectively. Three entomopathogenic nematodes strains of species Heterorhabditis bacteriophora were highly effective in killing wireworms (88 %, 53 %, and 53 % of mortality). The encapsulation of natural extracts in alginate beads appears to be a promising lure to manipulate wireworms behavior, while the co-encapsulation with entomopathogenic fungi and nematodes remains the major challenge for the development of a new Attract-and-kill strategyNEMACO

Arbuscular mycorrhizal fungi impact the production of alkannin/shikonin and their derivatives in Alkanna tinctoria Tausch. grown in semi-hydroponic and pot cultivation systems

IntroductionAlkanna tinctoria Tausch. is a medicinal plant well-known to produce important therapeutic compounds, such as alkannin/shikonin and their derivatives (A/Sd). It associates with arbuscular mycorrhizal fungi (AMF), which are known, amongst others beneficial effects, to modulate the plant secondary metabolites (SMs) biosynthesis. However, to the best of our knowledge, no study on the effects of AMF strains on the growth and production of A/Sd in A. tinctoria has been reported in the literature.MethodsHere, three experiments were conducted. In Experiment 1, plants were associated with the GINCO strain Rhizophagus irregularis MUCL 41833 and, in Experiment 2, with two strains of GINCO (R. irregularis MUCL 41833 and Rhizophagus aggregatus MUCL 49408) and two native strains isolated from wild growing A. tinctoria (R. irregularis and Septoglomus viscosum) and were grown in a semi-hydroponic (S-H) cultivation system. Plants were harvested after 9 and 37 days in Experiment 1 and 9 days in Experiment 2. In Experiment 3, plants were associated with the two native AMF strains and with R. irregularis MUCL 41833 and were grown for 85 days in pots under greenhouse conditions. Quantification and identification of A/Sd were performed by HPLC-PDA and by HPLC-HRMS/MS, respectively. LePGT1, LePGT2, and GHQH genes involved in the A/Sd biosynthesis were analyzed through RT-qPCR.ResultsIn Experiment 1, no significant differences were noticed in the production of A/Sd. Conversely, in Experiments 2 and 3, plants associated with the native AMF R. irregularis had the highest content of total A/Sd expressed as shikonin equivalent. In Experiment 1, a significantly higher relative expression of both LePGT1 and LePGT2 was observed in plants inoculated with R. irregularis MUCL 41833 compared with control plants after 37 days in the S-H cultivation system. Similarly, a significantly higher relative expression of LePGT2 in plants inoculated with R. irregularis MUCL 41833 was noticed after 9 versus 37 days in the S-H cultivation system. In Experiment 2, a significant lower relative expression of LePGT2 was observed in native AMF R. irregularis inoculated plants compared to the control.DiscussionOverall, our study showed that the native R. irregularis strain increased A/Sd production in A. tinctoria regardless of the growing system used, further suggesting that the inoculation of native/best performing AMF is a promising method to improve the production of important SMs

Arbuscular Mycorrhizal Fungi. Methods and Protocols.

Ferrol, N.; Lanfranco, L. (Eds.) 2020 Arbuscular Mycorrhizal Fungi. Methods and Protocols. Vol 2146. pp. 257. ISBN 978 1 0716-0602-

Long-term preservation of Arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) are obligate root symbionts, forming associations with most existing terrestrial plants. The plants obtain inorganic nutrients (e.g. N, P) via their fungal partners in exchange of which they provide the fungi with carbon compounds. AMF improve plant growth, health and productivity and as such, represent key organisms in agro-ecosystems. Currently, AMF diversity is maintained via continuous culture; in vivo on trap plants under greenhouse facilities, and in vitro in association with excised, transformed or nontransformed roots. However, these methods are time and work-consuming and the risks of contaminations and loss of genetic stability are not excluded. The objective of this thesis was to develop a long-term preservation method adapted to different AMF species and genera that guarantee their viability and stability over unlimited time of storage. In a first step, we tested different long-term preservation protocol (e.g. lyophilization, vitrification, cryopreservation) on Rhizophagus sp. MUCL 43204 produced in vitro. Cryopreservation appeared the most reliable method and was used in a second step on a large number of AMF isolates cultured in vitro. After 6 months storage at -130 °C, the viability of AMF isolates and their ability to colonize plant roots and reproduce the life cycle were tested. In a third step, we evaluated the morphology, physiological activity and genetic stability of 6 months-cryopreserved AMF isolates belonging to the genus Rhizophagus. Finally we applied the cryopreservation protocol developed on a large array of AMF isolates cultured either in vitro or in vivo. Our results demonstrated that the cryopreservation by encapsulation-drying and storage at -130°C of AMF propagules was effective, at least for 6 months, for different Rhizophagus isolates cultured in vitro. The method comprised five steps (1) the encapsulation in alginate beads of AMF propagules (i.e. spores and mycorrhizal root pieces) isolated from 5 months old cultures, (2) the incubation overnight in trehalose (0.5M), (3) the drying at 27°C for 2 days (i.e. at 8.1 ± 4.6% of beads water content), (4) the cryopreservation at -130 C in a freezer following a 2 steps decrease in temperature: a fast decrease (~12°C min-1) from room temperature (+20°C) to -110°C followed by a slow decrease in temperature (~1°C min-1) from -110°C to -130°C and (5) the direct thawing in a water bath set at +35°C. The morphology, physiological activity and genetic stability evaluated after 6 months storage at -130°C were similar to the non-cryopreserved controls. This method was also successfully applied to isolates belonging to Glomus, Claroideoglomus, Septoglomus, Paraglomus and Gigaspora cultured either in vitro or in vivo. Ours findings highlights the possibility to use the encapsulation-drying method for the cryopreservation and storage at -130°C of AMF isolates produced either in vitro or in vivo. These results further open the door to isolates that remain recalcitrant to different forms of long-term preservation at ultra-low temperature.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 201

Préservation à ultra-basse température des champignons mycorhizien à arbuscule cultivés in vitro par encapsulation-déshydratation.

À l'heure actuelle, plus de 300 espèces de champignons mycorhiziens à arbuscules (CMA) sont identifiés, dont la plupart sont maintenus dans des collections internationales. Ils sont conservés le plus souvent en serre sur des plantes pièges et, pour certains, in vitro en association avec des racines excisées. Ces méthodes de conservation demandent un travail considérable et, pour les premières, présentent un risque de contamination non négligeable. La culture in vitro des CMA en association avec des racines excisées représente une alternative aux problèmes de contaminations. Néanmoins, le risque de variation somaclonale pendant le processus de sub-culture ne peut être exclu. Dès lors, une méthode de préservation à long terme s’avère indispensables afin de garantir la conservation de ces microorganismes et leur stabilité génétique. Dans cette étude, douze souches de CMA cultivées in vitro en association avec des racines de carottes transformées ont été encapsulées dans des billes d'alginate et cryopréservées à ultra-basse température. Plusieurs protocoles ont été testés en tenant compte de l’âge de la culture, de l’agent cryoprotectant du status hydrique et du mode de refroidissement. La viabilité des souches de CMA a été estimée par le pourcentage de billes potentiellement infectieuses (% BPI), qui mesure le % de billes qui contiennent au moins une propagule germée. Le comportement thermique des billes d'alginate a été analysé par calorimétrie différentielle à balayage avant et après leur séchage pour estimer le pourcentage d’eau cristallisée et non cristallisée pendant le processus de cryoconservation. Il a été montré que les dommages des spores étaient directement liés à la cristallisation d’eau au cours de la cryopréservation. L'âge de la culture et l’agent cryoprotectant sont également des paramètres déterminants dans la cryopréservation de CMA. Quelle que soit la souche de CMA testée, la méthode optimale pour leur cryopréservation comprend 5 étapes: (1) l’encapsulation des propagules (spores et fragments de racines mycorhizés) isolées à partir de cultures âgées de 5 mois, (2) l’ incubation pendant une nuit dans une solution de tréhalose (0,5 M), (3) le séchage pendant 48h à 27 ° C, (4) la cryoconservation direct dans un congélateur à -130 ° C suivant deux étapes de refroidissement: une diminution rapide de température (~ 12 ° C min-1) de la température ambiante (20 ° C) jusqu’à -110 ° C, suivie d'une diminution lente (environ 1 ° C min-1) de -110 ° C à -130 ° C, et (5) un réchauffement direct à +35 ° C. Le PIB était supérieur à 70% pour toutes les souches et même au-dessus de 95% pour 11 des 12 souches après plusieurs mois de stockage à ultra-basse température. Toutes les souches ont conservé leur capacité d’associer in vitro des racines de carotte excisées et de reproduire leur cycle de vie avec la production de plusieurs centaines à milliers de spores après deux mois. Cette méthode ouvre la porte pour la conservation à ultra-basse température à long terme des souches de CMA dans des collections internationales

Préservation à ultra-basse température des champignons mycorhiziens à arbuscules: Impact sur la stabilité génétique et les propriétés physiologiques

A l’heure actuelle les champignons mycohriziens sont conservés essentiellement en culture continue sur plante piège en pot, pour la grande majorité des espèces, et en culture in vitro associée à des racines transformées, pour un nombre restreint d’espèces. Ces méthodes de conservation rend très difficile l’établissement de grande collection, elles demandent un travail considérable, beaucoup de main d’œuvre et présentent des risques de contamination et de variation somaclonale. Dès lors, des méthodes de préservation à long terme s’avèrent indispensables afin de garantir la conservation des espèces et leurs potentialités agro-environnementales. Plusieurs essais satisfaisants de préservation de spores de champignon mycorhizien à arbuscule ont été réalisés par le passé comme la lyophilisation ou le séchage et le stockage de spores de pots de cultures à 4°, cependant aucune de ces techniques ne peut garantir l’absence de microorganismes indésirables et la stabilité génétique du matériel conservé. Au cours des dernières décennies une attention particulière à été portée à la conservation des cellules végétales, animales et des microorganismes à ultra-basse température. En-dessous de -100°C, toutes les divisions cellulaires sont arrêtées et les événements métaboliques sont bloqués. Les cultures peuvent donc être conservées sans modification pendant une durée théoriquement illimitée à l’abri des contaminations et avec un entretien réduit. En 2000, Declerck et Van Coppennolle ont cryopréservés pour la première fois un Champignon mycorhizien à arbuscule produit en culture in vitro, les spores de Glomus intraradices encapsulées dans les billes d’alginates et cryopréservées à -130 °C ont conservé leur potentiel de germination et de colonisation des racines. Cependant ces résultats n’ont pas pu être reproduits pour d’autres souches ni sur de longues périodes de cryopréservation. Dans ce contexte, L’objectif général de notre travail est de développer une technique de cryopréservation à long terme, de l’appliquer aux différentes espèces de champignons mycorrhiziens à arbuscules cultivés in vitro et d’évaluer leur stabilité génétique et morphologique après cryopréservation. Dans un premier temps, l’objectif de cette étude est d’essayer le protocole d’encapsulation préservation sur une autre souche de Glomus intraradices et de voir l’effet de différent cryoprotectant et de l’age physiologique du champignon sur ça résistante à la cryopréservation

Preservation at ultra-low temperature of in vitro cultured Rhizophagus species with entrapment-dehydration

Aims and Background At present, 300 species of arbuscular mycorrhizal (AM) fungi have been identified, but their preservation under adequate conditions to ensure their long-term stability remains a challenge. The objective of this study was to demonstrate the possibility to cryopreserve these fungal symbionts. Methods From in vitro obtained spores, several parameters were considered for their cryopreservation: culture age, entrapment in alginate beads, cryoprotectant solutions, alginate beads drying and cooling rate. Thermal behaviour of alginate beads was analysed by differential thermal calorimeter before and after drying to estimate the frozen and unfrozen water during the cryopreservation process. Results It was shown that the spore damage was directly related to ice formation during cryopreservation. We also noticed that the culture age and cryoprotectant were determinant parameters for ultra-low temperature freezing success. Conclusion From these results, cryopreservation of 11 AM fungal strains was achieved following 5 step procedure: (1) alginate entrapment of spore from 5 months-old cultures, (2) overnight trehalose (0.5M) cryoprotectant treatment, (3) 48h drying at 27°C, (4) cryopreservation at -130°C with a - 0.25°C.min-1 cooling rate, and (5) direct thawing in water bath (+35°C). Whatever the strain considered, bead germination varied between 65-100% and all the strains kept their capacity to associate with a suitable host plant

Efficacy of entomopathogenic fungi against the fruit fly Drosophila suzukii and their side effects on predator and pollinator insects

peer reviewedAbstract Entomopathogenic fungi (EPF) are insecticide alternatives for pest control. Their ability to easily adhere and quickly penetrate the insect cuticle is a key factor for their selection, which has received too little consideration so far. Here, we evaluated the impact of five EPF on the survival and fecundity of Drosophila suzukii, a worldwide invasive pest of soft-skinned fruits. We assessed the exposure time needed to achieve fly mortality as well as the mortality of two non-target insects: Orius laevigatus and Bombus terrestris, commonly encountered in greenhouses where D. suzukii is the most damaging. Drosophila suzukii were exposed for 3 hours to a fungal culture from each EPF and survival rates were assessed daily. Beauvaria bassiana was the most efficient EPF, killing over 95% of the flies within 10 days. Additional flies were then exposed to this fungus culture for 10 seconds, 1 minute, 10 minutes and 1 hour. The exposure time impacted the mortality rates: 50% of the flies died within 4 days after a 3-hours exposure to B. bassiana, whereas 6 days were needed to reach the same result with 10 seconds of exposure. Whatever the exposure time, this EPF always needed ten days to be lethal for more than 95% of individuals. Beauvaria bassiana was not lethal for the non-target species. Thus, B. bassiana is an option to control D. suzukii without harming beneficial insects. Further studies are now needed under real cultivation conditions to assess whether B. bassiana can be included in biocontrol strategies against D. suzukii