Beltsville Agricultural Research Center

Abstract Vertical transmission of plant pathogenic viruses is an important component of viral persistence, survival, and spread in agricultural production systems. This type of transmission is of considerable economic significance as it can cause major crop losses by serving as the initial focus of infection for future epidemics. Vertical transmission occurs when a virus is passed on to offspring either by direct invasion of the developing seed embryo from infected mother plants or through infected pollen grains after fertilization. We have recently demonstrated by high throughput sequencing that mature seeds of the agriculturally important forage crop alfalfa ( Medicago sativa L.) are associated with a broad range of viruses some of which could potentially spread over long distances via seed. Aside from alfalfa mosaic virus, little is currently known about viral transmission via alfalfa pollen and its role in the epidemiology in this crop. This research was conducted to screen the pollen obtained from unique alfalfa genotypes for the presence of pathogenic viruses and their potential for dissemination. The plants from which the pollen was collected were alfalfa genotypes selected for fungal plant disease resistance and agronomic performance in a USDA ARS pre-breeding program in Prosser, WA.

<title>Abstract</title> As a nearly universal rule governing parasitology, parasites form aggregated distributions where a minority of hosts are responsible for harboring a majority of the parasite burden in any given population. For 30 years ectoparasitic <italic>Varroa destructor</italic> mites are described as preferring non-reproductive female nurse bees of its host, Western honey bee, <italic>Apis mellifera</italic> , while otherwise largely leaving the distribution of <italic>Varroa</italic> undescribed. As a result, <italic>Varroa-</italic> vectored disease transmission dynamics, sampling methodology and research have largely been worker centric. For the first time we investigate the intracolony distribution and dispersal patterns of <italic>Varroa</italic> on sex and age cohorts of its honey bee host in natural colonies and in laboratory choice experiments, and demonstrate that <italic>Varroa</italic> overwhelmingly prefer young adult males (drones), over any other cohort. We show <italic>Varroa</italic> form highly aggregated distributions on the drone cohort early in the season when infestation levels are low, and distribute broadly onto the worker cohort later in the season when infestation levels are high. In 1978 Anderson and May first proposed the theoretical framework that parasites would have little destabilizing effect on the host population until the degree of aggregation was low, or approached a Poisson distribution. The distribution patterns of <italic>Varroa</italic> are in alignment with their theoretical work, and maybe the underlying mechanism responsible for historical losses of honey bee colonies observed over the last decade.

Our protocol outlines a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay, which combines CRISPR/Cas12a technology with isothermal Recombinase Polymerase Amplification (RPA) for the rapid and specific detection of phytoplasma diseases in plants. This isothermal method utilizes RPA to amplify the target DNA fragment from the genomic DNA of phytoplasmas, followed by incubation with Cas12a nuclease and CRISPR RNAs (crRNAs) specifically designed to target unique phytoplasma DNA sequences. Upon initial cleavage of the amplified target DNA, Cas12a gains enzymatic activity to indiscriminately cleave single-stranded fluorescent oligonucleotide reporters, generating a fluorescent signal for highly sensitive detection of the pathogen. The protocol provides detailed instructions on: (i) sample collection and preparation; (ii) assay reaction setup, including RPA and Cas12a detection steps; (iii) reaction and detection conditions; and (iv) guidelines for accurately interpreting fluorescence data to detect phytoplasma DNA. This protocol is designed for researchers and agricultural professionals to effectively adopt and implement this advanced diagnostic technique.

The emergence of life on Earth likely involved a complicated evolution of the primeval residues via basic intermediate forms capable of self-replication. These primordial replicators could have further evolved into archaic virus-like structures, which in turn became the precursors of the cellular life forms. If viruses were indeed the predecessors of the first cellular life forms as suggested by the 'primordial virus world' and 'virus-first' scenarios, could their hosts themselves emerged and evolved predominantly as factories and reservoirs for virus production and dissemination? In other words, is that hypothetically possible that viruses were not only the originators of cellular life forms and the selfish driving force behind their evolution, but the fundamental reason for both their existence and biological heterogeneity? A short note presented here deliberates on this not entirely unfeasible course of events.