Retention and transmission of Grapevine leafroll-associated virus 3 by Pseudococcus calceolariae

Abstract

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically significant virus of grapevines found worldwide. The main mechanism of GLRaV-3 spread is by several species of mealybugs and soft-scale insects. Typically, mealybug abundance is linked to GLRaV-3 spread through a vineyard. An unusual finding in a New Zealand organic vineyard which had high mealybug populations yet no spread of GLRaV-3 prompted the hypothesis that viruliferous mealybugs feeding on non-virus hosts, such as groundcover plants, may lose GLRaV-3 and disrupt virus transmission. To test this hypothesis, it was first imperative to determine the timing of retention and transmission of GLRaV-3 by a mealybug vector, Pseudococcus calceolariae, after access to an alternate plant host (a non-virus host such as white clover). A highly sensitive one-step RT-qPCR TaqMan protocol was designed to detect GLRaV-3 in single first and second instar mealybugs, exuviae and grapevine tissue. The detection protocol detected as few as 10 GLRaV-3 RNA copies which enabled highly sensitive virus detection within mealybugs. Following a 5 day access acquisition period (AAP), P. calceolariae first instars retained GLRaV-3 for 4 days when fed on either an alternate host or GLRaV-3-free grape plants. In addition, GLRaV-3 was retained in some second instar and exuviae after feeding on GLRaV-3 positive grape leaves shortly before moult. GLRaV-3 has not previously been reported to be retained in P. calceolariae first instars for such a long period and GLRaV-3 has not previously been detected in the exuviae of first instar mealybugs and in second instar mealybugs. Following a 5 day AAP, GLRaV-3 was transmitted by P. calceolariae nymphs after access to white clover for up to 11 days. The 11 day retention period, followed by transmission of GLRaV-3, is the longest retention period observed in mealybugs vectoring GLRaV-3 to date. These results suggest that groundcover plants may act as a virus sink only if mealybugs settle and feed on them for several weeks. Based on retention of GLRaV-3 after moult, there is evidence to suggest GLRaV-3 is transmitted in a circulative manner; further experiments are required to verify this finding. This research project provides the foundations for future research to address the complex interactions between viruliferous mealybugs, grapevines, and nonvirus host plants. Such knowledge is fundamental for GLRaV-3 management in vineyards. Keywords: Pseudococcus calceolariae, Grapevine leafroll-associated virus 3, alternate host, retention, transmission