Transmission from seed to seedling and elimination of alfalfa viruses

Abstract

Introduction: Viral diseases have become a vital factor limiting the development of the alfalfa (Medicago sativa) industry. Six viruses infecting alfalfa with a high incidence rate are Alfalfa mosaic virus (AMV), Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa alphapartitivirus 2 (MsAPV2), Medicago sativa deltapartitivirus 1 (MsDPV1), Medicago sativa amalgavirus 1 (MsAV1), and Cnidium vein yellowing virus 1 (CnVYV1). The purpose of this study was to develop preventive measures against these viruses by investigating their transmission through alfalfa seeds.

Methods: In this study, we investigated the transmission rate of alfalfa viruses from seed to seedling by PCR, determined the location of viruses in seed by dissecting seed embryos and seed coat, tracked the changes of viruses in seedlings, and finally discover effective elimination measures for alfalfa viruses from 16 measures.

Results and discussion: Our results demonstrated that all these six viruses could be transmitted from alfalfa seeds to seedlings with the transmission rate ranging from 44.44% to 88.89%. For AMV, MsAPV2, and MsAV1, the viral load was significantly higher in the seed coats than in the seed embryos; however, it did not show significant differences between these two parts of the seeds for MsAPV1, MsDPV1, and CnVYV1. Dynamic accumulation analysis of AMV and MsAPV2 indicated that the viral load in plants increased continuously in the early growth stage, making it important to inactivate these viruses prior to their seed-to-seedling transmission. Sixteen treatments including physical, chemical, and combinations of physical and chemical measures were compared in terms of their elimination efficiency on AMV and MsAPV2 and impacts on seed germination. The results showed that soaking alfalfa seeds in sterile distilled water for 2h + 2% NaClO for 1h or 2% NaClO for 1h were more promisingly applicable because it could significantly reduce AMV and MsAPV2 particles in both seeds and seedlings. Our data revealed a route of virus transmission in alfalfa and shed light on the discovery of a highly efficient method for the management of alfalfa viral diseases.

Keywords: Medicago sativa alphapartitivirus 2 (MsAPV2); alfalfa mosaic virus (AMV); alfalfa viruses; transmission from seed to seedling; viruses elimination.

Figure 1

Heat map of the absolute amounts of six viruses detected in the seeds of 12 alfalfa cultivars. Virus copies were quantified by qRT-PCR. The logarithm of detected virus copies to base 10 is color coded according to the scale as shown. To ensure the representation of all samples, a value of 0.01 was assigned as the Ct value for the samples with no virus detected.

Figure 2

Virus titers comparison between the seed coats and embryos of alfalfa cv. “Zhongmu No.1”. Bar indicate the SE of virus concentration in four replicates, and letters “(A, B) and (a, b)” marks on the bar indicate significant differences between six treatments of coat and embryo, respectively (p < 0.05).

Figure 3

Dynamic accumulation of AMV (A) and MsAPV2 (B) in seedlings. Sampling time (X-axis) started from Day 1 when the first true leaves were unfolded. Different letters on top of error bars indicate significant (p < 0.5) differences of virus copy numbers.

Figure 4

Elimination of AMV (A) and MsAPV2 (B) from alfalfa seeds using nine different treatments (T1-T3, T10, T12-T16, refer to Table 2 for details) which effectively eliminated the viruses from alfalfa seeds and seedlings. Detected virus copies in the seedlings germinated from the treated seeds and in the treated seeds are shown on the left and right, respectively. Different uppercase or lowercase letters next to error bars indicate significant (p < 0.05) differences in viruses in seedling or seeds, respectively. “-” indicated the data unavailable in this treatment.

Figure 5

Germination energy and germination rate of alfalfa cv. “Zhongmu No.1” seeds disinfected using 16 different treatments (T1-T16, refer to Table 2 for details). Significant (p < 0.05) and extremely significant (p < 0.01) differences from the untreated seeds (CK) are indicated by single and double asterisks, respectively, shown on top of error bars.