Synthesis and insecticidal activities of novel solanidine derivatives.

BACKGROUND: Potato (Solanum tuberosum) is the fourth culture in the world and is widely used in the agri-food industries. They generate by-products in which α-chaconine and α-solanine, the two major solanidine-based glycoalkaloids of potato, are present. As secondary metabolites, they play an important role in the protection system of potato and are involved in plant protection against insects. To add value to these by-products, we described here new glycoalkaloids that could have phytosanitary properties. RESULTS: Solanidine, as a renewable source, was modified with an azido linker and coupled by copper-catalyzed alkyne azide cycloaddition to alkynyl derivatives of the monosaccharides found in the natural potato glycoalkakoids: D-glucose, D-galactose and L-rhamnose. The efficacy of our compounds was evaluated on the potato aphid Macrosiphum euphorbiae. The synthetic compounds have stronger aphicidal properties against nymphs than unmodified solanidine. They also showed strong aphicidal activities on adults and a negative impact on fecundity. CONCLUSION: Our synthetic neoglycoalkaloids affected Macrosiphum euphorbiae survival at the nymphal stage as well as at the adult stage. Furthermore, they induced a decrease in fecundity. Our results show that chemical modifications of by-products may afford new sustainable compounds for crop and plant protection. © 2018 Society of Chemical Industry.

A Secreted MIF Cytokine Enables Aphid Feeding and Represses Plant Immune Responses.

Aphids attack virtually all plant species and cause serious crop damages in agriculture. Despite their dramatic impact on food production, little is known about the molecular processes that allow aphids to exploit their host plants. To date, few aphid salivary proteins have been identified that are essential for aphid feeding, and their nature and function remain largely unknown. Here, we show that a macrophage migration inhibitory factor (MIF) is secreted in aphid saliva. In vertebrates, MIFs are important pro-inflammatory cytokines regulating immune responses. MIF proteins are also secreted by parasites of vertebrates, including nematodes, ticks, and protozoa, and participate in the modulation of host immune responses. The finding that a plant parasite secretes a MIF protein prompted us to question the role of the cytokine in the plant-aphid interaction. We show here that expression of MIF genes is crucial for aphid survival, fecundity, and feeding on a host plant. The ectopic expression of aphid MIFs in leaf tissues inhibits major plant immune responses, such as the expression of defense-related genes, callose deposition, and hypersensitive cell death. Functional complementation analyses in vivo allowed demonstrating that MIF1 is the member of the MIF protein family that allows aphids to exploit their host plants. To our knowledge, this is the first report of a cytokine that is secreted by a parasite to modulate plant immune responses. Our findings suggest a so-far unsuspected conservation of infection strategies among parasites of animal and plant species.

Solanidine isolation from Solanum tuberosum by centrifugal partition chromatography.

The aim of this investigation was the preparative isolation of solanidine (aglycone of the two main potato glycoalkaloids: α-chaconine and α-solanine) from fresh Solanum tuberosum (cv. Pompadour) material by implementing a new preparation scheme using centrifugal partition chromatography (CPC). A setup for obtaining solanidine by hydrolysis of the glycoalkaloids found in the skin and sprouts of S. tuberosum was first developed. Then its isolation was carried out by the development of CPC conditions: the solvent system used for separation was ethyl acetate/butanol/water in the ratio 42.5:7.5:50 v/v/v, 0.6 g of crude extract were separated with a 8 mL/min flow rate of mobile phase while rotating at 2500 rpm. A run yielded 98 mg of solanidine (86.7% recovery from the crude extract) in a one-step separation. The purity of the isolated solanidine was over 98%. Thus, CPC has proven to be the method of choice to get solanidine of very high purity from S. tuberosum biomass in large quantities.

Preparative separation of glycoalkaloids α-solanine and α-chaconine by centrifugal partition chromatography.

The main glycoalkaloids of a commercial potato cultivar, α-chaconine and α-solanine, were extracted from sprouts of Solanum tuberosum cv. Pompadour by a mixture of MeOH/H(2)O/CH(3)COOH (400/100/50, v/v/v). In these conditions, 2.8±0.62g of crude extract were obtained from 50g of fresh sprouts and the total glycoalkaloid content was determined by analytical HPLC at 216.5mg/100g. α-Chaconine and α-solanine were separated in a preparative scale using centrifugal partition chromatography (CPC). In a solvent system composed of a mixture of ethyl acetate/butanol/water (15/35/50, v/v/v), α-chaconine (54mg) and α-solanine (15mg) were successfully isolated from the crude extract in one step of purification. The purity of isolated compounds was determined to be higher than 92% by HPLC analysis.

Strong aphicidal activity of GlcNAc(ß1→4)Glc disaccharides: synthesis, physiological effects, and chitinase inhibition.

The synthesis of four GlcNAc(ß1→4)Glc disaccharides containing 2-O-acetyl and/or 6-sulfate groups was performed in high yields with total 1,2-trans stereoselectivity. These disaccharides were evaluated as candidates for insect chitinase inhibition and aphicidal activity. All the compounds prepared displayed physiological effects on M. persicae aphids; however, the inhibition of chitinases of different sources (bacteria, fungus, and aphid) followed different patterns according to subtle structural characteristics.

Purification and characterisation of a 31-kDa chitinase from the Myzus Persicae aphid: a target for hemiptera biocontrol.

Hydrolytic enzymes involved in chitin degradation are important to allow moulting during insect development. Chitinases are interesting targets to disturb growth and develop alternative strategies to control insect pests. In this work, a chitinase from the aphid Myzus persicae was purified with a 36-fold purification rate in a three step procedure by ammonium sulphate fractionation, anion-exchange chromatography on a DEAE column and on an affinity Concanavalin A column. The purified chitinase purity assessed by 1D and 2D SDS-PAGE revealed a single band and three spots at 31 kDa, respectively. Chitinases were found to have high homologies with Concanavalins A and B, two chitinase-related proteins, a fungal endochitinase and an aphid acetylhydrolase by peptide identification by Maldi-Tof-Tof. The efficiency of two potent chitinase inhibitors, namely allosamidin and psammaplin A, was tested and showed significant rate of enzymatic inhibition.

Assessing aphids potato virus Y-transmission efficiency: A new approach.

In order to develop an alternative method to optimize the relative efficiency factor (REF) assessment, the efficiency of transmission of Potato virus Y (PVY) by seven aphid species was examined. In vitro micropropagated potato plantlets were used to experiment on phenotypically and genetically homogeneous material. Species-specific acquisition access period (AAP) on a PVY-infected plantlet was assessed for each aphid species using electrical penetration graph (EPG) technique. Aphid probing behaviour determined by EPG showed that Macrosiphum euphorbiae and Myzus persicae exhibited the shortest AAPs (15 and 11min, respectively) whereas Rhopalosiphum padi, Sitobion avenae, Brevicoryne brassicae and Acyrthosiphon pisum exhibited the longest ones (more than 30min). The transmission rate obtained for M. persicae (83.3%) was higher than the ones reported in the literature. REFs assessment showed that A. pisum and B. brassicae were poor efficient vectors while M. euphorbiae and S. avenae seemed to be efficient ones even though their respective REF were significantly lower than that of M. persicae. The species R. padi and A. fabae did not transmit PVY. The hypothesis assessed for M. euphorbiae and S. avenae and consisting in the compensation of a weak PVY-transmission efficiency by a higher number of vectors, was not supported. The use of this new method for REF evaluation and the need to consider aphid behaviour for such an assessment was discussed.

A phloem-sap feeder mixes phloem and xylem sap to regulate osmotic potential.

Phloem-sap feeders (Hemiptera) occasionally consume the dilute sap of xylem, a behaviour that has previously been associated with replenishing water balance following dehydration. However, a recent study reported that non-dehydrated aphids ingested xylem sap. Here, we tested the hypothesis that the consumption of xylem sap, which has a low osmolality, is a general response to osmotic stresses other than dehydration. Alate aphids were subjected to different treatments and subsequently transferred onto a plant, where electrical penetration graph (EPG) was used to estimate durations of passive phloem sap consumption and active sucking of xylem sap. The proportion of time aphids fed on xylem sap (i.e., time spent feeding on xylem sap/total time spent feeding on phloem plus xylem sap) was used as a proxy of the solute concentration of the uptake. The proportion of time alate aphids fed on xylem sap increased: (1) with the time spent imbibing an artificial diet containing a solution of sucrose, which is highly concentrated in phloem sap and is mainly responsible for the high osmotic potential of phloem sap; (2) with the osmotic potential of the artificial diet, when osmotic potential excess was not related to sucrose concentration; and (3) when aphids were deprived of primary symbionts, a condition previously shown to lead to a higher haemolymph osmotic potential. All our results converge to support the hypothesis that xylem sap consumption contributes to the regulation of the osmotic potential in phloem-sap feeders.

Probing behavior of apterous and alate morphs of two potato-colonizing aphids.

Secondary host plant colonization by aphids involves alate and apterous morphs to spread in the population at a large scale by flying or, at a finer one, by walking. Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae) and Myzus persicae Sulzer (Hemiptera: Aphididae) are two polyphagous aphids that cause serious losses on many crops, particularly on potato, Solanum tuberosum L. (Solanales: Solanaceae). When settlement of virginoparous alate aphids occurs, apterous individuals are produced and spread within the potato field. As these two potato colonizers originate from different areas and show different body length, this study compared probing behaviors of virginoparous alate and apterous M. persicae and M. euphorbiae on one of their secondary host plants, Solanum tuberosum. Non­choice bioassays and electrical penetration graph (EPG) recordings were performed. Most M. euphorbiae of the two morphs rapidly accepted potato plants and exhibited long duration of probing, phloem sap salivation, and ingestion phases. In contrast, at the end of the experiment, most alates of M. persicae left the potato leaflet after brief gustative probes. Moreover, EPG experiments showed that the main difference between both morphs of the two species concerned the xylem ingestion parameter. Differences between species were also reported, such as an increased total duration of probing in both morphs and enhanced phloem ingestion duration in apterous M. euphorbiae. All the differences highlighted in this study are discussed according to the variations observed in aphid body size and to their historical association with Solanum species.

Compatible plant-aphid interactions: how aphids manipulate plant responses.

To access phloem sap, aphids have developed a furtive strategy, their stylets progressing towards sieve tubes mainly through the apoplasmic compartment. Aphid feeding requires that they overcome a number of plant responses, ranging from sieve tube occlusion and activation of phytohormone-signalling pathways to expression of anti-insect molecules. In addition to bypassing plant defences, aphids have been shown to affect plant primary metabolism, which could be a strategy to improve phloem sap composition in nutrients required for their growth. During compatible interactions, leading to successful feeding and reproduction, aphids cause alterations in their host plant, including morphological changes, modified resource allocation and various local as well as systemic symptoms. Repeated salivary secretions injected from the first probe in the epidermal tissue up to ingestion of sieve-tube sap may play a crucial role in the compatibility between the aphid and the plant.

Role of xylem consumption on osmoregulation in Macrosiphum euphorbiae (Thomas).

Aphids are phloem feeders that occasionally ingest xylem sap. The duration of xylem consumption by Macrosiphum euphorbiae (Hemiptera: Aphididae) was positively correlated with the level of dehydration of alate aphids of different ages after a period of starvation, supporting the hypothesis that aphids ingest xylem sap to replenish their water balance. However, the duration of xylem sap ingestion but not phloem sap consumption varied in unstarved alate adults of different ages. Furthermore, both alate and apterous aphids ingested xylem sap at the end of their life, when aphids were not dehydrated but when fecundity started to decrease. Fecundity was negatively correlated with the proportion of time spent ingesting xylem sap, and that over the entire reproductive life of alate and apterous aphids. The lower proportion of xylem ingested by apterous than by alate aphids during the first few days of adult life may be related to a higher symbiont density in apterous morphs. As previous studies have demonstrated a relationship between sucrose assimilation, which is directly influenced by fecundity and symbiont density, and osmoregulation, we suggest that xylem consumption may play a role in the osmoregulation of haemolymph of aphids.

Wild Solanum resistance to aphids: antixenosis or antibiosis?

The type (antixenosis or antibiosis) of resistance against the aphids Myzus persicae (Sulzer) and Macrosiphum euphorbiae (Thomas) was characterized for the wild tuber-bearing potatoes, Solanum chomatophilum Bitter and Solanum stoloniferum Schltdl. & Bouché through behavioral (olfactometry and electrical penetration graph) and physiological studies. In dual-choice assays, only S. stoloniferum exerted attraction to M. euphorbiae. This ruled out the possibility that plant volatiles of S. chomatophilum and S. stoloniferum may contribute to the high resistance expressed. In electrical penetration graph experiments, aphids feeding on S. stoloniferum showed increased salivation phases, whereas phloem ingestion was drastically reduced for both aphid species. Because reaching phloem elements was not delayed in both species, the resistance mechanism was phloem-located. The antixenosis exhibited by S. stoloniferum was similar on young and mature leaves. S. chomatophilum also showed phloem-located antixenosis against M. persicae. In contrast, M. euphorbiae had no difficulty to reach S. chomatophilum phloem tissues and to ingest sap. S. chomatophilum resistance against M. euphorbiae was antibiosis and only expressed in mature leaves, where a complete nymphal mortality was observed.

Differential aphicidal effects of chitinase inhibitors on the polyphagous homopteran Myzus persicae (Sulzer).

Four chitinase inhibitors, cyclo-(Proline-Tyrosine), cyclo-(Histidine-Proline), allosamidin and psammaplin A, were selected for in vitro feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Artificial diets were used to provide chitinase inhibitors at 10, 50 and 100 microg mL(-1) to M. persicae. Except for cyclo-(Proline-Tyrosine), which did not modify aphid demographic parameters, chitinase inhibitors induced differential aphicidal effects on M. persicae. At all doses, cyclo-(Histidine-Proline) induced significant effects affecting daily fecundity, intrinsic rate of natural increase (r(m)) and doubling time of population. When compared with the control diet, allosamidin decreased nymph survival and daily fecundity, increasing the doubling time of population from 1 to 1.5 days. Psammaplin A was the most toxic inhibitor when delivered via artificial diet, as it induced the death of all aphids reared at 50 and 100 microg mL(-1). The results demonstrate the potential use of chitinase inhibitors as aphid management tools.

Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linné) and in vitro.

With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid population's growth. Artificial diets were used to provide active (1, 10, 100 and 500 microg ml(-1)) and inactive (500 microg ml(-1)) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day population's doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.

Rapid method to screen resistance of potato plants against Myzus persicae (Homoptera: Aphididae) in the laboratory.

With the objective to develop a potato, Solanum tuberosum L., resistance program against aphids, we propose a rapid screening method with Myzus persicae (Sulzer) in the laboratory. We aimed to optimize the duration of the whole procedure and to decrease the frequency of measurements. In a first experiment, intrinsic rate of natural increase (r(m)) values were compared between adult aphids reared throughout their entire life and adults reared only during a period equivalent to their prereproductive period. No significant differences were observed. In a second experiment, four groups of aphids were distinguished according to the sampling frequency, i.e., those whose biological parameters were evaluated every single, second, third, and fourth day. Except for the fourth-day experiment, the r(m) values estimated on aphids reared on the three potato lines were not significantly different whatever sampling frequency of single, second, or third day used to check aphids. Thus, screening efforts in laboratory can be largely optimized by evaluating adult aphids only during a period equivalent to their prereproductive period and assessing M. persicae populations every third day. Our method is reliable and adapted to screen a large number of potato plants against M. persicae because it allows an average 70% reduction in the time required for the whole experimental process.

Comparative study of the strategies evolved by two parasitoids of the genus Asobara to avoid the immune response of the host, Drosophila melanogaster.

Asobara tabida and Asobara citri are two braconid endoparasitoids of Drosophila melanogaster larvae. We studied and compared the strategies evolved by these two species to avoid the immune reaction of their host. A. tabida has no negative impact on host cellular defenses and its eggs avoid encapsulation by adhering to host tissues. At the opposite, we found that A. citri, whose eggs are devoid of adhesive properties, affects the host encapsulation abilities, hemolymph phenoloxidase activity and concentrations of circulating hemocytes. Some of these effects could directly rely on a severe disruption of the hematopoietic organ anterior lobes observed in parasitized larvae. This is the first report of the immune suppressive abilities of a parasitoid from the Asobara genus. Results are presented and discussed with respect to the strategies of virulence evolved by other parasitoids to counteract the D. melanogaster immune system.