bioRxiv2021
Actinidia seed-borne latent virus (ASbLV, Betaflexiviridae), was detected at high frequency in healthy seedlings grown from lines of imported seed in a New Zealand post-entry quarantine facility. To better understand how to manage this virus in a dioecious crop species, we developed a rapid molecular protocol to detect infected progeny and to identify a reliable plant tissue appropriate to detect transmission rates from paternal and maternal parents under quarantine environment. The frequency of ASbLV detection from true infection of F1 progeny was distinguished by testing whole seeds and progeny seedling tissues from a controlled cross between two unrelated parents; an ASbLV-infected staminate (male) plant and an uninfected pistillate (female) plant, and the process was repeated with an ASbLV uninfected staminate (male) plant and an infected pistillate (female) plant. Individual whole seeds, or single cotyledons from newly-emerged seedlings, true leaf or a root from those positive-tested seedlings, were assessed for presence of ASbLV by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The virus was detected at a high incidence (98%) in individual seeds, but at a much lower incidence in seedling cotyledons (62%). Since detection results were consistent (P=95%) across the three seedling tissues (i.e. cotyledons, leaves and roots) only cotyledons were tested thereafter to determine ASbLV transmission to F1 progeny. F1 seedlings from three crosses were used to compare transmission rates from infected staminate versus infected pistillate parents. One cross from a single flower used an uninfected pistillate vine pollinated by an infected staminate vine, and two crosses (also from a single flower) used an infected pistillate vine (a sibling of the infected staminate vine), pollinated by either of two unrelated uninfected staminate vines. Cotyledon testing of seedlings from each cross confirmed staminate transmission at high frequency ([~]60%), and pistillate transmission at even higher frequency (81% and 86%, respectively). The results show ASbLV is transmitted at very high rates, whether from infected ovules or pollen. Transmission to seedlings is lower than detection in whole seeds perhaps due to ASbLV being sometimes residing on (or within) the seed coat only. The results also show RT-PCR of cotyledons allows non-destructive detection of ASbLV in very young seedlings, and could be used to screen kiwifruit plants in a nursery to avoid virus spread to orchards. Likewise, bulk testing of seed lots can quickly detect infected parent plants (fruit bearing female or male pollinator) already in an orchard. ImportanceActinidia seed-borne latent virus (ASbLV, Betaflexiviridae), was detected at high frequency in healthy seedlings grown from lines of imported seed in a New Zealand post-entry quarantine facility. However there are several technical barriers to detecting the presence of seed transmitted viruses and understanding their biology, which has significance for detection in quarantine and subsequent management under germplasm collections. To overcome this, we developed a rapid molecular protocol to detect infected progeny and to identify a reliable plant tissue appropriate to detect transmission rates from paternal and maternal parents under quarantine environment. Individual whole seeds, or single cotyledons from newly-emerged seedlings, true leaf or a root from those positive-tested seedlings, were assessed for presence of ASbLV by reverse transcription-polymerase chain reaction (RT-PCR) analysis. This was done with seed lots obtained from four separate controlled crosses between ASbLV-infected and ASbLV-uninfected Actinidia chinensis var. deliciosa parents.