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The Bemisia tabaci species complex threatens worldwide agriculture. While host-plant resistance is sustainable and effective, it is a relatively unexplored strategy for whitefly control. Three alfalfa ( Medicago sativa L.) populations were developed and used in high-throughput B. tabaci MEAM1 nymph-mortality screens. The phenotypic resistance/susceptibility spectrum in these populations indicated that whitefly resistance to MEAM1 is multigenic. Alfalfa lines highly resistant (R1, R2, and R3) and susceptible (S1) to B. tabaci MEAM1 were identified and further characterized. When life history parameters for three B. tabaci species (MEAM1, MED and NW1) were assessed on the four lines, whitefly species-specific responses to R1, R2 and R3 plants were revealed. While nymph mortality was high for MEAM1 and NW1 on resistant alfalfa, MED nymph development was surprisingly unimpaired. In addition, significant differences in oviposition, host-choice and longevity were observed amongst the three B. tabaci species. Collectively, these data indicated that R1, R2 and R3 plants each express a unique set of antibiotic/antixenotic resistance traits providing a potent multigenic and multi-faceted resistance to whiteflies with each B. tabaci species displaying distinct behaviors on the resistant lines. Our nymph-mortality simulation models indicate that deployment of the nymph mortality-mediated resistance of R1 and R3 alfalfa could substantially suppress whitefly population expansion in the field.

Background Aphids that feed on alfalfa ( Medicago sativa ) are widespread pests, causing significant losses in yield and forage quality through direct feeding damage and the transmission of plant viruses. In this study, we assessed aphid resistance in 239 genetically diverse alfalfa accessions across multiple field environments by quantifying aphid infestation levels. Results Leveraging more than 1.9 million high-quality single nucleotide polymorphisms, a genome-wide association study identified 22 genomic loci significantly associated with aphid resistance. These loci were distributed across all eight alfalfa chromosomes and collectively explained between 4.05% and 24.19% of the observed phenotypic variance. Functional annotation of the candidate genes revealed their involvement in key biological pathways, including ubiquitin-mediated proteolysis, ABC transporter activity, and immune signaling processes. Notably, two NLR-type resistance genes, Msa.H.0143490 and Msa.H.0461950 , emerged as strong candidates for enhancing aphid resistance through genetic improvement. Conclusions These findings provide valuable insights into the genetic architecture of aphid resistance and offer molecular markers that can be utilized in marker-assisted selection to develop more resilient alfalfa cultivars.

Nematodes are one of the major limiting factors in alfalfa production. Root-knot nematodes (RKN, Meloidogyne spp.) are widely distributed and economically important sedentary endoparasites of agricultural crops and they may inflict significant damage to alfalfa fields. As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests and specifically against RKN. In this work, we performed root transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and Lahontan, respectively. Bioinformatic analysis revealed a number of common and unique genes that were differentially expressed in susceptible and resistant lines as a result of nematode infection. Although the susceptible cultivar showed a more pronounced defense response to the infection, feeding sites were successfully established in its roots. Characteristically, basal gene expression levels under normal conditions differed between the two cultivars as well, which may confer advantage to one of the genotypes toward resistance to nematodes. Differentially expressed genes were subsequently assigned to known Gene Ontology categories to predict their functional roles and associated biological processes. Real-time PCR validated expression changes in genes arbitrarily selected for experimental confirmation. Candidate genes that contribute to protection against M. incognita in alfalfa were proposed and alfalfa-nematode interactions with respect to resistance are discussed.

Thrips (Thysanoptera: Thripidae) is a major insect pest for alfalfa which can result in decreased plant nutrients, low yields, and even plant death. To identify the differentially expressed genes and metabolites in response to thrips in alfalfa, a combination of metabolomics and transcriptomics was employed using alfalfa (Caoyuan No. 2) with and without thrips infestation. The results showed that the flavonoid biosynthesis and isoflavonoid biosynthesis pathways were the most significantly enriched pathways in response to thrips infection, as shown by the combined transcriptome and metabolome analysis. The transcriptome results showed that SA and JA signal transduction and PAPM-triggered immunity and the MAPK signaling pathway–plant pathways played a crucial role in thrips-induced plant resistance in alfalfa. In addition, we found that thrips infestation could also induce numerous changes in plant primary metabolism, such as carbohydrate and amino acid metabolism as compared to the control. Overall, our results described here should improve fundamental knowledge of molecular responses to herbivore-inducible plant defenses and contribute to the design of strategies against thrips in alfalfa.

Background Cystatins are cysteine protease inhibitor in plant and animal that not only regulates the intracellular transport of endogenous proteins in plants but also plays an important role in plant defense against biotic and abiotic stresses. However, the cystatin gene family in alfalfa remains poorly characterized. Result In this study, 55 cystatin genes distributed across 18 chromosomes in alfalfa were identified. GO enrichment analysis indicated that the 55 MsCYS genes were primarily associated with cysteine-type endopeptidase inhibitor activity. These genes were classified into six distinct subgroups, with each containing one or two typical cystatin domains. Analysis of cis -acting regulatory elements suggested the potential involvement of MsCYS genes in stress responses. Transcriptome analysis revealed that nearly half of the MsCYS genes were not expressed across the examined tissues and stress conditions and those that are expressed showed higher expression levels in stems and leaves than in roots. Expression levels of MsCYS genes were higher in stems and leaves than in roots. Under stress caused by thrips, MsCYS49 and MsCYS54 were significantly up-regulated. Real-time RT-PCR results corroborated the transcriptome data. The expression of MsCYS49 and MsCYS54 increased markedly, reaching 5.5-fold and 8.3-fold that of the control levels. After 14 days of thrips infestation. The cysteine content in alfalfa leaves increased significantly, which was associated with the up-regulation of MsCYS genes. Additionally, exogenous application of jasmonic acid enhanced the expression of certain MsCYS genes, thereby conferring resistance to thrips in alfalfa. Exogenous salicylic acid also up-regulated the expression of certain MsCYS genes and modulated insect defense. Conclusion Our study identified 55 members of the cystatin gene family in alfalfa and analyzed their phylogenetic relationships, structural characteristics, and putative functions in resistance to biotic and abiotic stress. This work establishes a robust foundation for further functional characterization of each cystatin gene member, which provides novel insights for improving the stress tolerance of alfalfa.

The spotted alfalfa aphid (Therioaphis trifolii) is a kind of destructive pest of cultivated alfalfa (Medicago sativa) that results in significant financial losses for the livestock sector. To understand how T. trifolii navigates the biochemical defenses of its host, we investigated the effects of susceptible and resistant aphid strains on two alfalfa cultivars. T. trifolii was reared for over 50 generations on two cultivars—WL343, which is susceptible to T. trifolii, and Zhongmu No. 1, which is resistant—resulting in the development of a resistant aphid strain (R-aphid) and a susceptible aphid strain (S-aphid). The results showed that the survival rate of R-aphids was significantly higher than that of S-aphids (p = 0.039) on the resistant cultivar Zhongmu No. 1, while there was no significant difference in survival rates between the two aphid strains on WL343 (p = 0.139). This suggests that S- and R-aphids differ in their ability to modulate plant defense mechanisms, influencing their survival rates. The application of saliva from R-aphids reared on Zhongmu No. 1 (R-saliva) reduced the repellency and toxicity of treated plants, improving aphid survival. Furthermore, R-aphid infestation and R-saliva application activated the salicylic acid (SA) signaling pathway while suppressing the jasmonic acid (JA) pathway, enhancing the host plant’s suitability for aphid colonization. We propose that R-aphids may use their saliva to activate the SA pathway, which in turn inhibits JA synthesis, weakening the plant’s defenses. These findings provide valuable insights into how T. trifolii interacts with host plant defense systems.

The establishment of the fall armyworm (FAW), Spodoptera frugiperda , in Africa is reported to have caused substantial agricultural losses since its detection in 2016 and subsequent spread into Asia and Australia by 2020. Based on the crops being infested, it appears that the population (C strain) primarily responsible for FAW infestations of corn in the Americas is widespread in Africa but there is uncertainty about the status of the R strain that targets pastures, alfalfa, millet, and rice in the United States. The two strains can only physically be distinguished by molecular markers, with single nucleotide polymorphisms (SNPs) in the mitochondrial Cytochrome oxidase subunit I ( COI ) and nuclear Z-chromosome-linked Triosephosphate isomerase ( Tpi ) gene demonstrated to consistently identify strains in both American continents. However, the COI and Tpi markers are generally in disagreement in the Eastern Hemisphere. This together with conflicting results from whole genome SNP studies creates uncertainty about the strain composition of this invasive population. In this paper the legitimacy of the Tpi markers is supported and used to not only confirm the existence of the R strain in Africa, but to also provide evidence for the introduction of new R strain variants in western Africa since 2017. These findings have implications on the crops at risk in the Eastern Hemisphere and for understanding how the invasion of Africa by FAW occurred.

Symbiotic bacteria play a crucial role in the survival, development, and adaptation of aphids to environmental conditions. Buchnera aphidicola (Enterobacterales: Erwiniaceae), the obligate endosymbiont of aphids, is essential for their fitness, while facultative symbionts may provide additional ecological advantages under specific conditions. A comprehensive understanding of how these symbiotic relationships respond to different climatic environments is essential for assessing aphid adaptability and potential implications for biological control. The present study investigates the vital interactions between the obligate bacterial endosymbiont, Buchnera aphidicola , and four facultative bacterial endosymbionts ( Arsenophonus sp., Hamiltonella defensa , Serratia symbiotica , and Regiella insecticola ), in black cowpea aphid (BCA), in the context of different climate conditions. The BCA specimens were obtained from the leaves of the host plant, alfalfa, cultivated in three distinct climates: cold semi-arid, hot desert, and humid subtropical climates. The findings, as anticipated, indicated a pervasive prevalence of B. aphidicola in BCAs infesting alfalfa crops across all three climate types. In contrast, the BCAs of each climate type exhibited a distinct array of facultative symbionts. The highest number of facultative endosymbionts was exhibited by BCAs from the humid subtropical climate, followed by BCAs from the cold semi-arid climate, whereas none of them were detected in BCAs from the hot desert climate. Rigiella insecticola was not detected molecularly in any of the BCAs from the three climates. Following the eradication of the obligate symbiont Buchnera aphidicola by the antibiotic rifampicin in BCAs, the effects on three categories of parameters were assessed, including life cycle stages, reproductive traits, and external morphological characteristics of adults. The most significant adverse effects were observed in BCAs inhabiting hot desert followed by those inhabiting cold semi-arid climate; detrimental effects in BCAs of the humid subtropical climate were considerably less pronounced. The observed discrepancies in the parameters of BCAs from the humid subtropical climate can be attributed to the presence of a greater number of facultative symbionts, especially the presence of Serratia symbiotica (Enterobacterales: Yersiniaceae). Following the eradication of B. aphidicola , this facultative symbiont continues to complement the functions of B. aphidicola in the host’s survival. Conversely, the low presence of facultative symbionts in cold semi-arid climate or even their absence in hot desert climate exacerbates the negative effects of obligate symbiont eradication. These findings highlight the crucial role of symbionts in aphid biology across a spectrum of climatic conditions, and suggest that shifts in symbiotic relationships may modulate aphid fitness, which could have implications for biological control programs.

When insects harm plants, they activate relevant enzyme systems for defense, and changes in enzyme activity, to a certain extent, reflect the host plant’s ability to resist insect damage. Alfalfa leaf weevils ( Hypera postica Gyllenhal) are the main economic insect pest of alfalfa, which seriously affect its yield and quality. To clarify the effects of feeding induction by alfalfa leaf weevil larvae and adults on defense enzymes in alfalfa, ‘Zhongmu No. 1’ variety was used as the experimental material. Comprehensive correlation analysis and principal component analysis were used to evaluate the corresponding patterns of 12 physiological indicators of alfalfa induced by insect feeding of different densities. Results showed that after feeding induction by adult and larval alfalfa leaf weevils, total antioxidant capacity (T-AOC), malondialdehyde (MDA), phenylalanine ammonia-lyase (PAL), tyrosine ammonia lyase (TAL), lipoxygenase (LOX), chymotrypsin inhibitors (CI), trypsin inhibitor (TI), and jasmonic acid (JA) in the alfalfa leaves increased with increasing feeding time. However, activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and polyphenolic oxidase (PPO) in alfalfa leaves first increased and then decreased, showing a downward trend.

Insect chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals from air to olfactory receptors in the lymph of antennal chemosensilla. They may represent a new class of soluble carrier protein involved in insect chemoreception. However, their specific functional roles in insect chemoreception have not been fully elucidated. In this study, we report for the first time three novel CSP genes (AlinCSP1-3) of the alfalfa plant bug Adelphocoris lineolatus (Goeze) by screening the antennal cDNA library. The qRT-PCR examinations of the transcript levels revealed that all three genes (AlinCSP1-3) are mainly expressed in the antennae. Interestingly, these CSP genes AlinCSP1-3 are also highly expressed in the 5(th) instar nymphs, suggesting a proposed function of these CSP proteins (AlinCSP1-3) in the olfactory reception and in maintaining particular life activities into the adult stage. Using bacterial expression system, the three CSP proteins were expressed and purified. For the first time we characterized the types of sensilla in the antennae of the plant bug using scanning electron microscopy (SEM). Immunocytochemistry analysis indicated that the CSP proteins were expressed in the pheromone-sensitive sensilla trichodea and general odorant-sensitive sensilla basiconica, providing further evidence of their involvement in chemoreception. The antennal activity of 55 host-related semiochemicals and sex pheromone compounds in the host location and mate selection behavior of A. lineolatus was investigated using electroantennogram (EAG), and the binding affinities of these chemicals to the three CSPs (AlinCSP1-3) were measured using fluorescent binding assays. The results showed several host-related semiochemicals, (Z)-3-hexen-1-ol, (E)-2-hexen-1-al and valeraldehyde, have a high binding affinity with AlinCSP1-3 and can elicit significant high EAG responses of A. lineolatus antennae. Our studies indicate the three antennae-biased CSPs may mediate host recognition in the alfalfa plant bug A. lineolatus.