Pathogen spread rates are determined, in part, by the performance of pathogens under altered environmental conditions and their ability to persist while switching among hosts and vectors.

To determine the effects of new conditions (host, vector, and nutrient) on pathogen spread rate, we introduced a vector-borne, viral plant pathogen, Barley Yellow Dwarf Virus PAV (BYDV-PAV) into hosts, vectors, and host nutrient supplies that it had not encountered for thousands of viral generations. We quantified pathogen prevalence over the course of two serial inoculations under the new conditions. Using individual level transmission rates from this experiment, we parameterized a dynamical model of disease spread and projected spread across host populations through a growing season.     

A change in nutrient conditions (increased supply of phosphorus) reduced viral transmission whereas shifting to a new vector or host species had no effect on infection prevalence.  However, the reduction in the new nutrient environment was only temporary; infection prevalence recovered after the second inoculation.

Synthesis. These results highlight how robust the pathogen, BYDV-PAV, is to changes in its biotic and abiotic environment. Our study also highlights the need to quantify longitudinal infection information beyond snapshot assessments to project disease risk for pathogens in new environments.

Natal conditions

Our experiment used BYDV-PAV viral cultures that had been maintained in the laboratory (see Viral Culture Source in Appendix) using a single aphid species, S. avenae, on a single host species, A. sativa, under low nutrient conditions for 251 days (see detailed information regarding vector colony and host plant source in Appendix, Vector Conditions and Host Conditions).  Aphid population growth under these natal conditions produces approximately 12 generations of S. avenae (Dedryver et al., 1998), and approximately 1,800 to 6,000 generations of BYDV-PAV (Yarwood, 1956). 

Treatment conditions

We experimentally exposed natal viral cultures to a range of conditions consisting of two aphid vectors, two plant hosts, and four nutrient conditions for a total of 16 treatments. The treatments consisted of a full cross of vector (two conditions), host (two conditions), and nutrient (four conditions) across the natal, S. avenae, or new aphid vector, R. padi, the natal, A. sativa, or new host species, H. vulgare, and the nutrient conditions.  The nutrient conditions included nitrogen (NH₄NO₃), phosphorus (KH₂PO₄), nitrogen plus phosphorus, or no additional nutrients.  Each treatment was repeated eight times over three consecutive temporal blocks for a total 24 replicates per treatment.  For each block of the experiment, 70 seeds from each host plant species were planted for a total of 140 plants.  Each block had eight replicates per treatment with 12 additional seeds planted to account for the possibility of for failed germinations.  The plants were watered with the four nutrient treatments: nanopure water only (Control), 10% nitrogen solution, 10% phosphorus solution, or 10% nitrogen & phosphorus solution all based on a Half-Strength Hoagland’s solution (Hoagland and Arnon 1950) which are consistent with previous experiments (Lacroix, Seabloom and Borer, 2014).  The first inoculation that introduced the virus to new biotic and abiotic conditions will be referred to as Round 1.  We then performed a second inoculation (referred to as Round 2) in vivo such that all treatments were applied to a set of hosts that maintained the treatment where plant tissue from Round 1 treatments was used to infect the aphids used in Round 2.  A simple schematic depicting the inoculation and treatment conditions is represented in Fig. 1.  Assessing viral evolution and population dynamics after serial passages is not uncommon (Sylvester, Richardson and Frazier, 1974; Kurath and Palukaitis, 1990; Schneider and Roossinck, 2000; Bartels et al., 2016) but quantifying virus transmission in serially passaged viruses after switching abiotic or biotic conditions has rarely been performed.  All plant tissues were collected between June 5th, 2017 and October 10, 2017.  All plant tissue was collected and preserved at -20C for molecular processing. 

Virus inoculation

Each block as described under Treatment Conditions, included two inoculation rounds.  During the first inoculation (Round 1) of the experiment, 360 live, adult-sized aphids of both R. padi and S. avenae were removed from uninfected plants and transferred to 25ml cork sealed tubes (24x) each containing 30 aphids of the same species.  Leaf tissue from approximately four-week-old plants confirmed to be infected with BYDV-PAV was clipped and 4-6 cm of infected tissue was transferred into each tube containing non-viruliferous aphids. Aphids remained in cork sealed tubes for 48 hours such that they became viruliferous from feeding on infected plant tissue, meaning the aphids were then able to transmit the virus. After 48 hours, aphids were moved to uninfected plants for the initial inoculation period.  Plants used for the initial inoculation were uninfected prior to aphid exposure as the plants remained isolated from aphids and other insects and there is no evidence of vertical transmission of BYDV-PAV in hosts. 

We controlled for factors known to influence transmission efficiency including length of feeding period on infected tissue and age of host tissue (Gray et al., 1991).  To do this, a single 2.5 x 8.5 cm, 118 μm polyester mesh cage was attached to the oldest leaf on each 17-day old experimental plant. Five viruliferous aphids were transferred into each polyester mesh cage which was then sealed.  The experimental plants containing the caged aphids were then placed in a growth chamber and aphids fed for approximately 96 hours, after which the aphids were killed to end transmission. 

At the start of the second inoculation (Round 2), the experimental plants from Round 1 were destructively harvested and the polyester mesh cages were removed.  Each 8.5 cm leaf enclosed by the cage was cut from the plant and transferred to a clean 25 ml tube while the above-ground plant tissue was stored at -20C.  Once all tissues were collected at the end of the experiment, BYDV-PAV infection status (presence/absence) was assessed using polymerase chain reaction (see Virus detection in Appendix).  Ten aviruliferous aphids (i.e., not yet carrying a virus) of each species were collected from vector source conditions, transferred into each of the tubes respective to the treatment, and allowed to feed for 48-hours.  Five aphids from each tube were transferred to a new experimental plant raised under the treatment conditions and cage-sealed; the remaining five aphids were discarded.  During Round 2, the treatments with R. padi in the sixth block only contained one aphid per cage due to R. padi colony depletion.  All other treatments contained five aphids per cage per block.  The experimental plants were subjected to feeding period of 96 hours after which the aphids were killed.  BYDV-PAV infection status (presence/absence) was assessed using polymerase chain reaction (see Virus detection in Appendix).