Genetic diversity, population structure, and taxonomic confirmation in annual medic (Medicago spp.) collections from Crimea, Ukraine.

Annual medic (Medicago spp.) germplasm was collected from the Crimean Peninsula of Ukraine in 2008 to fill gaps in geographic coverage in the United States department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS) temperate-adapted forage legume collection. A total of 102 accessions across 10 Medicago species were collected. To assess genetic diversity, population structure, and to confirm taxonomic identities, the collections were phenotypically and genetically characterized. Phenotyping included the use of 24 descriptor traits while genetic characterization was accomplished using a 3K Diversity Array Technologies (DArTag) panel developed for alfalfa (Medicago sativa L.). For both field and molecular characterizations, a reference set of 92 geographically diverse and species-representative accessions were obtained from the NPGS collection. Phenotypic descriptors showed consistency among replicated plants within accessions, some variation across accessions within species, and evident distinctions between species. Because the DArTag panel was developed for cultivated alfalfa, the transferability of markers to the species being evaluated was limited, resulting in an average of ~1,500 marker loci detected per species. From these loci, 448 markers were present in 95% of the samples. Principal component and phylogenetic analysis based on a larger set of 2,396 selected markers clustered accessions by species and predicted evolutionary relationships among species. Additionally, the markers aided in the taxonomic identity of a few accessions that were likely mislabeled. The genotyping results also showed that sampling individual plants for these mostly self-pollinating species is sufficient due to high reproducibility between single (n=3) and pooled (n=7) biological replicate leaf samples. The phenotyping and the 2,396 Single Nucleotide Polymorphism (SNP) marker set were useful in estimating population structure in the Crimean and reference accessions, highlighting novel and unique genetic diversity captured in the Crimean accessions. This research not only demonstrated the utility of the DArTag marker panel in evaluating the Crimean germplasm but also highlighted its broader application in assessing genetic resources within the Medicago genus. Furthermore, we anticipate that our findings will underscore the importance of leveraging genetic resources and advanced genotyping tools for sustainable crop improvement and biodiversity conservation in annual medic species.

Composition of the alfalfa pathobiome in commercial fields.

Through the recent advances of modern high-throughput sequencing technologies, the "one microbe, one disease" dogma is being gradually replaced with the principle of the "pathobiome". Pathobiome is a comprehensive biotic environment that not only includes a diverse community of all disease-causing organisms within the plant but also defines their mutual interactions and resultant effect on plant health. To date, the concept of pathobiome as a major component in plant health and sustainable production of alfalfa (Medicago sativa L.), the most extensively cultivated forage legume in the world, is non-existent. Here, we approached this subject by characterizing the biodiversity of the alfalfa pathobiome using high-throughput sequencing technology. Our metagenomic study revealed a remarkable abundance of different pathogenic communities associated with alfalfa in the natural ecosystem. Profiling the alfalfa pathobiome is a starting point to assess known and identify new and emerging stress challenges in the context of plant disease management. In addition, it allows us to address the complexity of microbial interactions within the plant host and their impact on the development and evolution of pathogenesis.

Characterization of the seed virome of alfalfa (Medicago sativa L).

BACKGROUND: Seed transmission of plant viruses can be important due to the role it plays in their dissemination to new areas and subsequent epidemics. Seed transmission largely depends on the ability of a virus to replicate in reproductive tissues and survive during the seed maturation process. It occurs through the infected embryo or mechanically through the contaminated seed coat. Alfalfa (Medicago sativa L.) is an important legume forage crop worldwide, and except for a few individual seedborne viruses infecting the crop, its seed virome is poorly known. The goal of this research was to perform initial seed screenings on alfalfa germplasm accessions maintained by the USDA ARS National Plant Germplasm System in order to identify pathogenic viruses and understand their potential for dissemination. METHODS: For the detection of viruses, we used high throughput sequencing combined with bioinformatic tools and reverse transcription-polymerase chain reactions. RESULTS: Our results suggest that, in addition to common viruses, alfalfa seeds are infected by other potentially pathogenic viral species that could be vertically transmitted to offspring. CONCLUSIONS: To the best of our knowledge, this is the first study of the alfalfa seed virome carried out by HTS technology. This initial screening of alfalfa germplasm accessions maintained by the NPGS showed that the crop's mature seeds contain a broad range of viruses, some of which were not previously considered to be seed-transmitted. The information gathered will be used to update germplasm distribution policies and to make decisions on the safety of distributing germplasm based on viral presence.

Snake River alfalfa virus, a persistent virus infecting alfalfa (Medicago sativa L.) in Washington State, USA.

Here we report an occurrence of Snake River alfalfa virus (SRAV) in Washington state, USA. SRAV was recently identified in alfalfa (Medicago sativa L.) plants and western flower thrips in south-central Idaho and proposed to be a first flavi-like virus identified in a plant host. We argue that the SRAV, based on its prevalence in alfalfa plants, readily detectable dsRNA, genome structure, presence in alfalfa seeds, and seed-mediated transmission is a persistent new virus distantly resembling members of the family Endornaviridae.

Diversity of the virome associated with alfalfa (Medicago sativa L.) in the U.S. Pacific Northwest.

Alfalfa (Medicago sativa L.) is one of the most extensively cultivated forage legumes in the world. It is currently the third most valuable field crop in the United States with an estimated value of over $9.3 billion. Alfalfa productivity is limited by various infectious diseases that can reduce forage yield and quality and shorten stand life. The crop can frequently be infected with a diverse array of pathogens and other organisms that have distinct life cycles, biology, and mode of action. Among them are many coinfecting viruses, that greatly contribute to the heterogeneity of within-host pathogenic communities, representing a ubiquitous and abundant background for all other host-pathogen interactions. Regrettably, the impact of viral diseases, their role in alfalfa health and involvement in the severity of multi-pathogen infections are often underestimated and not well understood. As high-throughput sequencing approaches have been developed, opportunities to delve into these complex interactions can be realized. In this work, we have characterized a diversity of viral populations in several commercial alfalfa production fields located in the U.S. Pacific Northwest. At least 45 distinct viruses have been identified in all alfalfa samples. Among them some were known to infect the crop prior to this study, and others were designated as emerging, novel and viruses integrated into the alfalfa genome. Known viruses included alfalfa mosaic virus, pea streak virus and bean leafroll virus, while among emerging and novel agents were alfalfa virus S, cherry virus Trakiya, several rhabdoviruses and others. Additional biological and impact studies will be needed to determine if newly identified viruses, especially those that have not been reported from alfalfa before, should be considered pathogens of this crop.

Attraction of Pollinators to Atemoya (Annona squamosa × Annona cherimola) in Puerto Rico Using Commercial Lures and Food Attractants.

Atemoya is a hybrid between Annona squamosa L. and Annona cherimola Miller (Annonaceae) and has potential to be an important fruit crop in tropical and subtropical areas. A major impediment to fruit production is low fruit set due to inadequate pollinator visits, typically, by beetles in the family Nitidulidae. We used Universal moth traps to monitor the attractiveness of two commercially available Nitidulidae lures in combination with various food attractants, including raw bread dough, apple juice, and malta beverage, a soft drink by-product of the brewing process. The most commonly trapped beetles were, in order of decreasing frequency, Carpophilus dimidiatus (F.), Brachypeplus mutilatus Erichson, Urophorus humeralis (F.) (Coleoptera: Nitidulidae), and Europs fervidus Blatchley (Coleoptera: Monotomidae). All traps, except the unbaited control traps, caught beetles. In a previous study, we found that combining two commercial lures had a synergistic effect on the attraction of these beetle species. In this study, the addition of food attractants increased the number of beetles trapped compared with traps baited with only the commercial lures. Also, food attractants appear to be key in attracting U. humeralis; only one U. humeralis individual of the 206 caught during the experiment was trapped without a food attractant. The variation between the number of beetles caught in traps containing the same treatments was high and may explain the erratic results reported in other studies of pollination in Annona spp. The results are discussed with respect to the use of nitidulid lures and food attractants to increase fruit set in atemoya and other Annonaceae.

Development of a Large Set of Microsatellite Markers in Zapote Mamey (Pouteria sapota (Jacq.) H.E. Moore & Stearn) and Their Potential Use in the Study of the Species.

Pouteria sapota is known for its edible fruits that contain unique carotenoids, as well as for its fungitoxic, anti-inflammatory and anti-oxidant activity. However, its genetics is mostly unknown, including aspects about its genetic diversity and domestication process. We did high-throughput sequencing of microsatellite-enriched libraries of P. sapota, generated 5223 contig DNA sequences, 1.8 Mbp, developed 368 microsatellites markers and tested them on 29 individuals from 10 populations (seven wild, three cultivated) from Mexico, its putative domestication center. Gene ontology BLAST analysis of the DNA sequences containing microsatellites showed potential association to physiological functions. Genetic diversity was slightly higher in cultivated than in the wild gene pool (HE = 0.41 and HE = 0.35, respectively), although modified Garza-Williamson Index and Bottleneck software showed evidence for a reduction in genetic diversity for the cultivated one. Neighbor Joining, 3D Principal Coordinates Analysis and assignment tests grouped most individuals according to their geographic origin but no clear separation was observed between wild or cultivated gene pools due to, perhaps, the existence of several admixed populations. The developed microsatellites have a great potential in genetic population and domestication studies of P. sapota but additional sampling will be necessary to better understand how the domestication process has impacted the genetic diversity of this fruit crop.

Attraction of pollinators to atemoya (Magnoliales: Annonaceae) in Puerto Rico: a synergistic approach using multiple nitidulid lures.

Atemoya, a hybrid between Annona squamosa (L.) and A. cherimola Miller (Annonaceae), has potential to be a major fruit crop in tropical and subtropical areas. A major setback to fruit production throughout the world is low fruit-set because of inadequate pollinator visits, typically Nitidulidae beetles. We identified beetle visitors to atemoya flowers in an orchard in Puerto Rico and used Universal moth traps to monitor the attractiveness of two commercially available Nitidulidae lures. The most common visitors to atemoya flowers were an unidentified Europs species (Coleoptera: Monotomidae), followed by Loberus testaceus (Coleoptera: Erotylidae), neither of which have been previously reported as visitors to Annona flowers. The commercial lures attracted few or no beetles when used separately, but attracted a large number of beetles, especially Carpophilus dimidiatus (Coleoptera: Nitidulidae) and Europs, when used in combination. This attraction is synergistic and increases with dose at the doses assayed (0-4 lures), and decreases over time with >50% of trap captures occurring in the first week and no beetles collected after 5 wk. This is the first report of aggregation pheromone lures in nitidulids acting synergistically to attract other species, including beetles not in the Nitidulidae. The results are discussed as they pertain to increasing fruit set, as well as the potential for altering fruit size and shape in atemoya.

Development of microsatellite loci in Artocarpus altilis (Moraceae) and cross-amplification in congeneric species.

PREMISE OF THE STUDY: Microsatellite loci were isolated and characterized from enriched genomic libraries of Artocarpus altilis (breadfruit) and tested in four Artocarpus species and one hybrid. The microsatellite markers provide new tools for further studies in Artocarpus. • METHODS AND RESULTS: A total of 25 microsatellite loci were evaluated across four Artocarpus species and one hybrid. Twenty-one microsatellite loci were evaluated on A. altilis (241), A. camansi (34), A. mariannensis (15), and A. altilis × mariannensis (64) samples. Nine of those loci plus four additional loci were evaluated on A. heterophyllus (jackfruit, 426) samples. All loci are polymorphic for at least one species. The average number of alleles ranges from two to nine within taxa. • CONCLUSIONS: These microsatellite primers will facilitate further studies on the genetic structure and evolutionary and domestication history of Artocarpus species. They will aid in cultivar identification and establishing germplasm conservation strategies for breadfruit and jackfruit.

Effect of coconut palm proximities and Musa spp. germplasm resistance to colonization by Raoiella indica (Acari: Tenuipalpidae).

Although coconut (Cocos nucifera L.) is the predominant host for Raoiella indica Hirst (Acari: Tenuipalpidae), false spider mite infestations do occur on bananas and plantains (Musa spp. Colla). Since its introduction, the banana and plantain industries have been negatively impacted to different degrees by R. indica infestation throughout the Caribbean. Genetic resistance in the host and the proximity of natural sources of mite infestation has been suggested as two of the main factors affecting R. indica densities in Musa spp. plantations. Greenhouse experiments were established to try to determine what effect coconut palm proximities and planting densities had on R. indica populations infesting Musa spp. plants. Trials were carried out using potted Musa spp. and coconut palms plants at two different ratios. In addition, fourteen Musa spp. hybrid accessions were evaluated for their susceptibility/resistance to colonization by R. indica populations. Differences were observed for mite population buildup for both the density and germplasm accession evaluations. These results have potential implications on how this important pest can be managed on essential agricultural commodities such as bananas and plantains.