1.11.15
MECHANICAL TRANSMISSION AND ISOLATION OF THE HIGH PLAINS PATHOGEN.

R LOUIE1 and DL SEIFERS2

1USDA-ARS and OARDC, The Ohio State University, Wooster, OH 44691, USA; 2Kansas State University and ARCH, KA 67601, USA

Background and objectives
A disease of maize (Zea mays L.) and wheat (Triticum aestivumL.) with virus-like symptoms has been widespread in the High Plains of the central and western USA in the past several years [1]. Symptoms of the high plains disease (HPD) in both hosts often begin as chlorotic spots that coalesce to form streaks and general chlorosis which are followed by necrosis in the affected areas of lower leaves. Seedling death is reported in highly susceptible genotypes. In some years, HPD posed a serious economic threat to both crops. The high plains pathogen (HPP) and wheat streak mosaic virus (WSMV) often are found together in the same plant. Five species of dsRNA and a 32 kD protein have been associated with diseased plants but not with an infectious agent. Both HPP and WSMV are transmitted by Aceria tosichella Keifer, but no pure culture of HPP has yet been continuously maintained by mite transmission. Mixed infections of WSMV and HPP in field grown and inoculated plants have confounded research efforts to determine the etiology and pathogenic effects of the disease. Hence, an ability to efficiently transmit HPP without mites and maintain it in pure culture would greatly facilitate the resolution of these problems.

Vascular puncture inoculation (VPI) of seeds successfully transmitted maize viruses that were previously obligately transmitted by vectors [2]. The transmission of maize and rice viruses by VPI presently includes: MCDV, MCMV, MDMV, MMV, MRFV, MRCV, MRDV, MSV, MStpV, MSMV, MWLMV, RBSDV, RHBV, SCMV, and WSMV [2 and unpublished data]. The premise for VPI is that for a limited time during the process of germination, the vascular system of an embryo is susceptible to mechanical inoculation. Recently we reported the transmission of HPP by VPI. The objective of this study was to improve the transmission rates of HPP with VPI and to isolate and maintain a pure culture of HPP.

Results and conclusions
The isolate was confirmed as HPP by positive reactions in enzyme-linked immunosorbent assay (ELISA) and Western blot with antiserum to HPP and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein analysis for HPP. The absence of WSMV was confirmed by an absence of symptoms in rub-inoculated maize (Oh28), wheat (var. Monon), and sorghum (Sorghum bicolor(L.) Moench var. ĎAtlas') and a negative reaction in ELISA and Western blot. High rates of transmission (90-100%) were achieved from extracts of infected, physiologically young tissues (ca 21 days after inoculation) and from the supernatant or pellet after low and high speed differential centrifugation. Lower rates of transmission (ca 75%) were obtained from the pellets after Cs2SO4 equilibrium density-gradient centrifugation. SDS-PAGE analysis associated infectivity with the 32 kD protein that is indicative of HPP infections. A culture of HPP has been maintained free of WSMV contamination in over 25 serial transfers.

HPP is thought to be similar to the pathogens of rose rosette, thistle mosaic, fig mosaic, redbud yellow ringspot, and wheat spot mosaic [1]. However, for these diseases, no pathogen has yet been isolated and no particle has been seen in partially purified preparations. HPP has been associated with double membrane bodies (150-200 nm in dia) often seen in transmission electron microscopy of fixed thin sections of infected leaves. Although we have demonstrated an infectious agent from plants infected with HPP by mechanical transmission with VPI, we have neither isolated nor observed the pathogenic agent.

References
1. Jensen SG, Lane LD, Seifers DL 1996. Plant Disease 80:1387-1390.
2. Louie R 1995. Phytopathology 85:139-143.