2013 Annual Report
1a.Objectives (from AD-416):
To develop and validate a universal plant virus microarray for detection and differentiation of plant viruses. To demonstrate the ability of an oligonucleotide microarray to detect and differentiate plant viruses from random amplification of plant total nucleic acid extracts.
1b.Approach (from AD-416):
ARS will acquire the lists of viral taxa to be represented on the viral detection microarray, and virus-infected samples from which to amplify nucleic acids to validate the microarray. This information and material will be utilized by both ARS and the Cooperator to jointly develop and validate the microarray for detection of target viruses, and to make validation results available to collaborators via a web server. The Cooperator will perform analysis of viral sequences to identify suitable sequences for the development of oligonucleotides, provide the facilities for production of the microarrays based on the selected oligonucleotides,and participate in analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples.
The Universal Plant Virus Microarray (UPVM) had been successfully used to detect and identify different virus species present in high titer without the need for multiple-rounds of amplification. To date, the number of virus species hybridized is small in comparison to the number of known plant virus species (>1300). In order to improve validation and performance of the UPVM and increase its utility to potential users, we obtained plant virus samples from different genera and hosts from national and international research institutes, and have investigated new technologies to enrich the amount of virus nucleic acid versus plant nucleic acid. Additional sets of virus species were obtained under a USDA-APHIS diagnostic permit as follows: (1) Grapevine field samples, Washington State University (WSU; Prosser. WA); (2) Fruit tree and hop samples, the National Clean Plant Network (NPCN)-Fruit tree (WSU; Prosser, WA); (3) Tospovirus samples (WSU, Pullman, WA); (4) Potato samples, USDA-ARS (Prosser, WA); and,(5) Viruses of cereals, USDA-ARS (Wooster, OH). Further samples were obtained from: (1) the International Potato Center (CIP) Lima, Peru (as potato and sweet potato samples from greenhouse, gene bank, field, and from alternate hosts); (2) the German Collection of microorganisms and cell culture (DSMZ) at Braunschweig, Germany; and, (3) the International Center for Tropical Agriculture (CIAT) Cali, Colombia (novel viruses from cassava).
In a pilot study of the samples from DSMZ, the UPVM successfully detected 21 viruses out of 56 samples without amplification, and one sample by modified ABC amplification. Potato yellowing virus was identified only by Ilarvirus genus level probes, as species-level probes for this virus are not present in the UPVM probe set.
In this period we have used 280 UPVM slides to hybridize samples, some of which belong to previously untested genera, including species of Benyvirus, Luteovirus, Nucleorhabdovirus, and Hostuviroid. We have also continued work on increasing the detection of low titer virus(es) by a modified ‘ABC amplification’ method, and have successfully detected Potato leaf roll virus (in a mixed infection potato sample from the CIP genebank) and Arracacha virus B (DSMZ). The modified ABC amplification method will be further optimized to increase detection rate and used for low titer samples including fruit tree samples obtained from NCPN, Prosser, WA.
In addition to changes in sample preparation and labeling, statistical methods (T-predict, Clustering, Z-score analysis) were employed to increase the detection rate. Using hierarchical clustering of healthy and infected sample hybridization profiles, three maize viruses were detected; these three samples were negative by the standard visual intensity method. Additional samples will be obtained from various sources in order to hybridize representatives of all possible plant virus genera and families. We have also prepared 2000 poly-L-lysine coated glass slides in order to print 750-1000 additional UPVM slides. This information will be of value to the UPVM collaborators, regulatory agencies, plant diagnostic clinics, germplasm repositories, and producers operating plant certification schemes.