**10. Diagnosis of tobamoviruses in seeds**

A technique for the detection of plant viruses that relies on the serological method enzymelinked immunosorbent assay (ELISA) [148–150] was adapted successfully for the detection of tobamoviruses in seeds. ELISA is considered to be a robust technique [151], and it enables the detection of viral CP subunits [152–159]. However, ELISA suffers from two main limitations: false-negative and false-positive results. The specificity and avidity of the antibodies used for the analysis can vary and may lead to false results. The International Seed Federation (ISF) http://www.worldseed.org/, the International Seed Testing Association (ISTA) https://www. seedtest.org/en/seed-health-methods-\_content---1--1452.html, and International Seed Health Initiative for Vegetable Crops (ISHI-Veg) http://www.worldseed.org/our-work/phytosanitary-matters/seed-health/ishi-veg/#protocols adapted, developed, and validated protocols for pathogen diagnosis in seed stock materials (**Table 2**). For the tobamoviruses such as TMV, ToMV, and PMMoV or the *Potexvirus Pepino mosaic virus* (PepMV) that infect *Solanaceae*, proper testing requires 12 batches of 250 seeds each (3000 seeds in total), with detection threshold of 1:249 (1 infected seed/249 healthy seeds). For detection of cucurbit-infecting viruses such as the *Tobamovirus* CGMMV, the *Melon necrotic spot virus* (MNSV, *Carmovirus*), and *Squash mosaic virus* (SqMV, *Comovirus*), and for the detection of the PSbMV (*Potyvirus*) and PEBV (*Chlorovirus*) infecting pea seeds, 20 batches of 100 seeds each (2000 seeds in total) are required. For the latter virus species that belong to five genera (CGMMV, MNSV, SqMV, PSbMV, and PEBV), ELISA detection threshold of 1:99 (1 infected seed/99 healthy seeds) was reported and according to ISTA: "The theoretical detection limit is one seed in 100. To ensure a 95% probability that infestations of 0.15% or higher are detected, it is necessary to test 20 subsamples of 100 seeds." The efficacy of a steadfast seed diagnosis relies on several parameters: (i) random collection of seed samples from the homogenous seed lot and (ii) seed disinfection treatments that are calibrated and examined in parallel to germination assays. In commercial seed production,


**Table 2.** Recommended diagnostic ratios for seed-contamination tests.

**9. Seed disinfection treatments**

are also applied in large-scale production.

are preserved in the seeds (**Figure 3**(**d–e**)) [34].

**10. Diagnosis of tobamoviruses in seeds**

Na3 PO<sup>4</sup>

246 Advances in Seed Biology

Methods that are used in large-scale commercial seed production are mostly based on various

and the most commonly used in commercial seed production 10% trisodium phosphate (TSP)

However, the recent global outbreaks of *Tobamovirus* diseases emphasize the incompetence of conventional seed disinfection treatments, which could be explained by the preservation of the virus in the inner tissues of the seeds [5]. Analyzing the efficacy of sequential treatments of 10% TSP followed by 72 h heat treatment at 72°C showed that CGMMV infectious particles

A technique for the detection of plant viruses that relies on the serological method enzymelinked immunosorbent assay (ELISA) [148–150] was adapted successfully for the detection of tobamoviruses in seeds. ELISA is considered to be a robust technique [151], and it enables the detection of viral CP subunits [152–159]. However, ELISA suffers from two main limitations: false-negative and false-positive results. The specificity and avidity of the antibodies used for the analysis can vary and may lead to false results. The International Seed Federation (ISF) http://www.worldseed.org/, the International Seed Testing Association (ISTA) https://www. seedtest.org/en/seed-health-methods-\_content---1--1452.html, and International Seed Health Initiative for Vegetable Crops (ISHI-Veg) http://www.worldseed.org/our-work/phytosanitary-matters/seed-health/ishi-veg/#protocols adapted, developed, and validated protocols for pathogen diagnosis in seed stock materials (**Table 2**). For the tobamoviruses such as TMV, ToMV, and PMMoV or the *Potexvirus Pepino mosaic virus* (PepMV) that infect *Solanaceae*, proper testing requires 12 batches of 250 seeds each (3000 seeds in total), with detection threshold of 1:249 (1 infected seed/249 healthy seeds). For detection of cucurbit-infecting viruses such as the *Tobamovirus* CGMMV, the *Melon necrotic spot virus* (MNSV, *Carmovirus*), and *Squash mosaic virus* (SqMV, *Comovirus*), and for the detection of the PSbMV (*Potyvirus*) and PEBV (*Chlorovirus*) infecting pea seeds, 20 batches of 100 seeds each (2000 seeds in total) are required. For the latter virus species that belong to five genera (CGMMV, MNSV, SqMV, PSbMV, and PEBV), ELISA detection threshold of 1:99 (1 infected seed/99 healthy seeds) was reported and according to ISTA: "The theoretical detection limit is one seed in 100. To ensure a 95% probability that infestations of 0.15% or higher are detected, it is necessary to test 20 subsamples of 100 seeds." The efficacy of a steadfast seed diagnosis relies on several parameters: (i) random collection of seed samples from the homogenous seed lot and (ii) seed disinfection treatments that are calibrated and examined in parallel to germination assays. In commercial seed production,

 mentioned above [5, 143]—which have been reported to provide satisfactory control of tobamoviruses in cucurbit [144] and solanaceous seeds (e.g., pepper [5, 145, 146] and tomato [147]). In addition to the chemical treatments, several heat treatment protocols at various temperature conditions ranging from 72 to 76°C for a minimum of 12 h up to 72 h [144]

, 1–3%

)2 ,

CSN(CH<sup>3</sup>

)2 NCSS<sup>2</sup>

chemical treatments—1–9% hydrochloric acid HCl, 1–5% calcium hypochlorite Ca(OCl)<sup>2</sup>

sodium hypochlorite NaOCl, tetramethylthiuram disulfide (TMTD) (CH<sup>3</sup>

applying conventional chemical disinfection treatments followed by extensive washes, which wash out the virus from the seed coat and decrease considerably the viral titer in the tested samples [145, 146], may end up in reducing the virus titer below the detection threshold. Seed analysis under these conditions will show false-negative results because viable viral particles still exist in the internal seed tissues, e.g., the perisperm-endosperm envelope in cucurbit seeds [34] and the endosperm in *Solanaceae* seeds [4, 35]. The second limitation of the ELISA method, which is false-positive results, may occur in instances that show lack of correlation between the ELISA results and biological significance. The possibility of obtaining false-positive or false-negative ELISA results may lead to situations in which growers purchase treated seed lots that are contaminated. Since the ELISA assay detects only the CP subunit, it is not possible to draw a direct link between ELISA results and the status of the viral particles or the degree of infectivity de facto. It is a major obstacle even when more sensitive molecular-based methods such as reverse transcription polymerase chain reaction (RT-PCR) [3, 160, 161] or quantitative real-time polymerase chain reaction (qRT-PCR) [162–164] are applied, which detect amplified partial genome fragments. Therefore, especially when dealing with treated/disinfected commercial seed lots, the preferable scenario is to validate the ELISA-positive seed subsamples in biological assays on susceptible indicator plants in order to ensure the infectivity status of the tested seed lot before marketing or sowing.
