Graduate Students Receive Grants from Northeast SARE

Juan Francisco Iturralde-Martinez
Ultrasensitive and On-field Detection of a Plant Virus by a Nanotube-filtering Device and Isothermal Amplification

Working with viruses can be challenging due to their nanometric size, the difficulty or impossibility of culture and the lack of specialized techniques for virus discovery. Moreover, the current methods of isolation require considerable time effort and the use of expensive equipment. CNT-STEM (Carbon Nanotube Size-Tunable Enrichment Microdevice) is a microfluidics device that allows the entrapment of viruses via physical sieving into vertically aligned carbon nanotubes and then their subsequent retrieval and analysis¹. This device is also useful for the discovery of new viruses, since no previous knowledge of the particles is needed other than their size.

As a proof of concept, Tomato spotted wilt orthotospovirus (TSWV) was proposed as a candidate virus to undergo CNT-STEM purification, because the virus can achieve high titers in planta, its spherical particles are in the range of action of the device, and because of its economic importance as plant pathogen. Recombinase Polymerase Amplification (RPA) is a molecular technique similar to PCR that is gaining more support for on-site pathogen detection, since the reaction can occur at room temperature, reducing the need for expensive and non-portable equipment for diagnostics. We propose coupling the physical sieving and concentration of viruses by CNT-STEM with the downstream use of RPA for on-site ultrasensitive detection of TSWV in early stage of infection.

¹Yeh, Y. T., Tang, Y., Sebastian, A., Dasgupta, A., Perea-Lopez, N., Albert, I., Terrones, M., Zheng, S. Y. (2016). Tunable and label-free virus enrichment for ultrasensitive virus detection using carbon nanotube arrays. Sci Adv, 2(10), e1601026. doi:10.1126/sciadv.1601026

Phillip Martin
Combining Infection Sources, Periods, and Persistence into the Integrated Management of Bitter Rot on Apples

Growing apples in the humid northeastern US is challenging due to fungal diseases like bitter rot, caused by fungi in the genus Colletotrichum. Historically a southern disease, northeastern apple growers are reporting increased problems with bitter rot. While the basic bitter rot disease cycle was discovered over 100 years ago, much remains unknown about inoculum sources and spore dispersal periods. We therefore propose to use the highly sensitive quantitative (q) PCR pathogen detection technique to identify and quantify infection sources and timing of dispersal in and around orchards, focusing on apple buds, the tree canopy, the orchard floor, and nearby wooded areas. It is well known that infected apples from one year can be a source of spores the next year unless the infected apple is degraded by other microbes. The degradation of infected apples on the orchard floor and reduction of Colletotrichum will be measured with q-PCR. The successional microbes (bacteria and fungi) that presumably drive that degradation will be identified with metagenomic amplicon sequencing and their physiological profiles obtained with Biolog EcoPlates, comparing the weed-free tree row with grass drive rows. Information on infection sources will inform cultural practices that reduce disease pressure and the need for heavy fungicide applications. Knowing the timing of critical infection periods will inform targeted and judicial applications of control products. This research will be combined with related studies on bitter rot fungal species and their fungicide sensitivity profiles to help northeastern apple growers improve their integrated management of bitter rot.

Joseph Walls
Exploring Disease Pressure in Response to Climate Change

Plant disease pressure is likely to increase with climate change, which necessitates research on novel methods of sustainable disease management. In the Northeast United States temperatures are rising along with shorter winters and more stochastic weather events that will have major effects on pest populations, many of which are vectors of pathogens. In turn, there is expected to be an increase in crop disease pressure with these climate changes. Here we propose to study the applicability of phytohormones for management of Tomato spotted wilt virus (TSWV) under climate change conditions. Jasmonic acid, salicylic acid, and abscisic acid will be applied to plants two days before and after inoculation with TSWV. These applications will also be tested under various temperatures and drought stress conditions. Disease incidence and severity will be measured two weeks post-inoculation. Results from the study will guide further research into manipulating these hormonal pathways to manage TSWV as well as other diseases. The findings of the proposed study will be presented at the American Phytopathological Society meeting and the Pennsylvania Association for Sustainable Agriculture Farming for the Future annual meeting as well as other grower meetings.