In PVY of potatoes, several strain groups are distinguished according to the symptoms on Nicotiana tabacum, Physalis floridana, Solanum tuberosum and other potato cultivars (4). The main groups are: the common (PVYO), tobacco veinal necrosis (PVYN), and stipple streak (PVYC) strain groups of PVY. More recently, subgroups within the historical groups have been described. For example within the PVYO group, atypical isolates, originating mostly from pepper and tobacco, have been assigned to the subgroup PVYOb, whereas the common European potato isolates are called PVYOa (8). Within PVYN, subgroups are represented by more recently described isolates such as PVYNTN (involved in the potato tuber necrotic ringspot disease) (9) and the Wilga type PVYNW (2). The latter one (PVYNW) is biologically similar to the «old» N-type (inducing typical necrosis on tobacco), but serologically similar to the O-type (no reaction with our «PVY-necrotic» reagent).
Details of detection of these isolates with our PVY reagents are given below in the section specificity and are illustrated in Table 1. BIOREBA AG has collaborated since 1980 with the Swiss Federal Research Station Agroscope Changins-Wädenswil (ACW, Nyon), and other institutes elsewhere for the development of ELISA reagents (7, 8). These collaborations allow us to adjust our reagents to new knowledge and new findings as needed. In this regard, we, in cooperation with the ACW, were able to modify and thus optimize our full-spectrum PVY reagent («PVY monoclonal cocktail») introduced many years ago (for details see below).
We rely as much as possible on monoclonal antibodies (Mab) for our reagents, since Mab provide several advantages over polyclonal antibodies (Pab): The reagent based on Mab guarantees a well defined and standardized product (constant in specificity and avidity), whereas Pab-based reagents inevitably introduce some variability between batches originating from different animals. Once developed, Mab are reproducible year after year. Different complementary Mab can be mixed in a controlled manner in order to broaden its serological range as it has been done for example with our «PVY monoclonal cocktail».
This approach using Pab may lead to increased non-specific reactions. On the other hand, Mab are suitable for differentiating PVYN subgroups (example described in section D below). Pathogen-specific Mab do not contain antibodies against host plant proteins that might interfere in the test. Therefore, non-specific background in ELISA is generally decreased, rendering the test more sensitive (wider OD ratio between infected and healthy tissue). But there are exceptional cases where non-specific reactions with Mab do occur (non-specific reactions with certain plant components?).
Our reagents have been extensively tested in Europe and at the International Potato Center (CIP) in Peru and were found to be very efficient (W. Bitterlin and P. Gugerli, unpublished; COST 88 PVY ring-test, unpublished; 5, 7, 10). Furthermore, they have been used since the early 1980’s by many scientists and in numerous potato certification laboratories in many parts of the world.
A. The traditional broad-spectrum PVY reagent («PVY monoclonal»), made against isolate PVYN605 and on the market since 1982, recognizes isolates belonging to the tobacco veinal necrosis (PVYN), common (PVYO) and stipple streak (PVYC) strain groups of PVY (7). The reagent is based on a monoclonal antibody (Mab) to a common antigenic determinant. All of today’s known PVY isolates are recognized by this Mab with the exception of a few atypical isolates (e.g. PVYO768) belonging to subgroup PVYOb (8). This reagent also recognizes PVYNTN(involved in the potato tuber necrotic ringspot disease) (9) and the Wilga type PVYNW (2). The reagent has proven its validity over the past quarter of a century in variable testing set-ups all over the world. As the first monoclonal-based reagent that became commercially available for phytodiagnostics, it really has become a true PVY standard reagent.
B. The full-spectrum PVY reagent («PVY monoclonal cocktail«) contains complementary monoclonal antibodies to isolates from different strain groups (7, 8). It recognizes the whole range of PVY isolates from the tobacco veinal necrosis (PVYN), common (PVYO) and stipple streak (PVYC) strain groups of PVY (Table 1). All recently described isolates such as PVYO768 (belonging to subgroup PVYOb) (8) or PVYNTN (involved in the potato tuber necrotic ringspot disease) (9) and the Wilga type PVYNW (2) are also recognized. This reagent generally gives a more uniform reaction with the virus and a lower background than polyclonal antibodies from rabbit or sheep serum. The current reagent that became available in 2002 is an improved version of the reagent introduced in 1994. It consists of a «cocktail» of different complementary monoclonal antibodies. No cross-reaction with any other known potato virus has been observed.
C. The polyclonal PVY reagent («PVY polyclonal«) was made against isolate PVYN605. It recognizes, similar to «PVY monoclonal cocktail», the whole range of isolates from the tobacco veinal necrosis (PVYN), common (PVYO) and stipple streak (PVYC) strain groups of PVY, including the more recently described isolates such as PVYO768 (belonging to subgroup PVYOb) (8) or PVYNTN (involved in the potato tuber necrotic ringspot disease) (9) and the Wilga type PVYNW (2). But contrary to the monoclonal-based reagent, some variability from batch to batch in recognizing isolates of different strain groups is inevitable with the polyclonal-based reagent.
D. The necrotic strain group-specific PVY reagent («PVY necrotic») specifically recognizes isolates belonging to the tobacco veinal necrosis (PVYN) strain group of PVY («old» N-type group), including subgroup of PVYNTN (involved in the potato tuber necrotic ringspot disease) (9). In an independent study, carried out in 1991 (COST 88 PVY ring-test, unpublished), where nearly 50 PVY isolates from Europe and other continents were compared in biological and serological tests, all isolates of PVYN (old N-type group) were recognized with our PVYN reagent. On the other hand, Wilga type PVYNW isolates (2) are not recognized (PVYNW isolates are recognized with our other PVY reagents, i.e. «PVY monoclonal», «PVY monoclonal cocktail», and «PVY polyclonal» described in sections A, B and C).
Our PVY reagents thus provide a suitable tool for differentiating PVYN subgroups (M. Chrzanowska, personal communication, and 2). The PVYN reagent is based on monoclonal antibodies, developed against PVYN605 (7, 8). The coating reagent consists of broad-spectrum, the conjugate of N-specific Mab (7). The PVYN reagent has been developed for double antibody sandwich ELISA (DAS-ELISA) (3, 6). This format is essential to ensure the described N-specificity.
