Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): detection of the AMV CP and npt2 transgenes in seeds, herbage and hay

White clover (Trifolium repens L.) is an important pasture legume in temperate areas throughout the world, providing fodder for grazing animals and improving soil fertility via symbiotic nitrogen fixation. However, the persistence and stress tolerance of white clover is affected by several viruses, chiefly Alfalfa mosaic virus (AMV), Clover yellow vein virus (ClYVV) and White clover mosaic virus (WClMV). Efforts to introgress natural forms of virus resistance from other Trifolium spp. into white clover and lucerne (alfalfa) have had only limited success. This has been addressed by developing white clover germplasm exhibiting viral-coat-protein-mediated resistance to AMV and non-transgenic resistance to ClYVV. This report describes PCR-based assays for detecting the transgenes associated with the H6 transformation event in seeds, fresh leaves, air-dried leaves and mixtures of air-dried herbage of white clover and perennial ryegrass (hay). Although further development is required to convert these assays for use in the field, this paper demonstrates the ability to detect these transgenes in a range of agricultural products associated with the commercial use of white clover.

Posttranscriptional Gene Silencing Does Not Play a Significant Role in Potato virus X Coat Protein-Mediated Resistance

The expression of a gene that encodes coat protein (CP) of Potato virus X (PVX) in transgenic tobacco plants confers a high level of CP-mediated rresistance (CP-MR) against PVX infection. To determine if posttranscriptional gene silencing (PTGS) plays a role in resistance, transgenic plants expressing PVX CP were challenged against PVX under conditions in which PTGS was suppressed by low temperatures or using viruses carrying PTGS suppressors. The data demonstrate that PTGS does not play a significant role in PVX CP-MR.

Coat–protein–mediated resistance to tobacco mosaic virus: discovery mechanisms and exploitation

In 1986 we reported that transgenic plants which accumulate the coat protein of tobacco mosaic virus (TMV) are protected from infection by TMV, and by closely related tobamoviruses. The phenomenon is referred to as coat–protein–mediated resistance (CP–MR), and bears certain similarities to cross protection, a phenomenon described by plant pathologists early in this century. Our studies of CP–MR against TMV have demonstrated that transgenically expressed CP interferes with disassembly of TMV particles in the inoculated transgenic cell. However, there is little resistance to local, cell–to–cell spread of infection. CP–MR involves interaction between the transgenic CP and the CP of the challenge virus, and resistance to TMV is greater than to tobamoviruses that have CP genes more distantly related to the transgene. Using the known coordinates of the three–dimensional structure of TMV we developed mutant forms of CP that have stronger inter–subunit interactions, and confer increased levels of CP–MR compared with wild–type CP. Similarly, it is predicted that understanding the cellular and structural basis of CP–MR will lead to the development of variant CP transgenes that each can confer high levels of resistance against a range of tobamoviruses.

The Association of Severe Epidemics of Cucumber Mosaic in Commercial Fields of Pepper and Tobacco in North Florida with Inoculum in Commelina benghalensis and C. communis

Since 1995, severe epidemics of cucumber mosaic virus (CMV) have occurred in select fields of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) in three counties in northern Florida. Yield losses greater than 50% have occurred in both crops. Baker and Zettler (1) identified the presence of CMV in one plant of tropical spiderwort (Commelina benghalensis) in an organic garden on the campus of the University of Florida 10 years ago. In addition, they infected tropical spiderwort and Asiatic dayflower (Commelina communis) with isolates of CMV. Since 1995, in one area of northern Alachua County, Asiatic dayflower has been found in abundance in and around some fields and found to be infected with CMV. Prior to this time, CMV had not been known to be epidemic in any crop in northern Florida. Also, commelinaceous weeds did not occur in such abundance in northern Florida. In Hamilton County, an epidemic of CMV occurred in one field of tobacco in 1997. Tropical spiderwort with viral-like symptoms was growing abundantly in that field. The symptoms in this weed included chlorotic ringspots and chevron-like line patterns. Light microscopy, with Azure A stain, revealed the presence of typical inclusions of CMV in pepper, tobacco, tropical spiderwort, and Asiatic dayflower. Symptomatic samples of the tobacco and the tropical spiderwort reacted in an immunodiffusion test with antiserum to a winged bean isolate of CMV (2). Extracts from tropical spiderwort (isolate 3603) were rubbed on squash. This isolate was thereafter maintained in squash (Cucurbita pepo cvs. Prelude II or Early Prolific Straightneck). Infected plants of both of these cultivars developed strong mosaic symptoms and were stunted. After passage through squash, the 3603 isolate induced mosaic in tobacco (cv. Burley 21). Some plants of the squash cultivars Destiny III and Liberator III, which have transgenic, coat protein-mediated resistance to CMV, developed restricted symptoms after inoculation with this isolate. CMV was recovered by back inoculation from symptomatic plants of these cultivars. Symptomless plants of tropical spiderwort transplanted from the field developed chlorotic ringspots and chevron-like line patterns following inoculation in the greenhouse with isolate 3603. Back inoculations to squash followed by immunodiffusion assays confirmed the presence of CMV in the inoculated tropical spiderwort plants but CMV was not detected in noninoculated control plants. This is the first report of tropical spiderwort being infected with CMV in a commercial situation in the United States. Because commelinaceous plants are well known to be excellent hosts of CMV (1), we believe that the increased presence of perennial, commelinaceous weeds is a factor contributing to the epidemics of CMV in northern Florida. References: (1) C. A. Baker and F. W. Zettler. Plant Dis. 72:513, 1988. (2) C. A. Ku-wite and D. E. Purcifull. Plant Dis. 66:1071, 1982.