First Report of Citrus viroid V Naturally Infecting Grapefruit and Calamondin Trees in California.

Abstract

The Citrus Clonal Protection Program–National Clean Plant Network, University of California, Riverside (UCR), received citrus budwood samples in June 2016 from the California Department of Food and Agriculture (CDFA) for virus and viroid testing under the mandatory California (CA) §3701 Citrus Nursery Stock Pest Cleanliness Program. Samples originated from nonsymptomatic budwood sources, two redblush grapefruit (RG) (Citrus paradisi Macfadyen) and four variegated calamondin (VC) (C. madurensis Lour.), and were found in a nursery in Tulare County, CA. TRIzol-extracted RNA was tested with a SYBR green I reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the universal detection of citrus viroids (Osman et al. 2017). Amplicons with melting temperatures similar to the Citrus viroid V (CVd-V) control (Vidalakis and Wang 2013) were produced in 50% of the RG and 25% of the VC samples. Citron (C. medica L.) Arizona 861-S-1 indicators were graft-inoculated with RG and VC blind buds (two citrons each) and maintained under warm greenhouse conditions (27 to 41°C). By 8 months, all citrons had developed mild stunting and leaf bending coupled with midvein necrosis, indicative of viroid infection. Sequential polyacrylamide gel electrophoresis of the symptomatic citrons identified circular and linear viroid-like RNAs with electrophoretic mobility similar to CVd-V. RT-PCR with primers specific for the full CVd-V genome (Wang et al. 2013) produced amplicons of the expected size of 294 bp from both the RG and VC samples and from the four indicator citrons. Cloning and sequencing of the RT-PCR CVd-V amplicons produced complete viroid genome sequences (GenBank MF477857 to MF477876). The RG isolates (MF477857 to MF477866) had 99% similarity with the KM isolate (JQ348924, Pakistan), with one nucleotide change (A₆₅ > U). The VC isolates (MF477867 to MF477876) had 99% similarity with the MO isolate (JQ348925, Pakistan), with two nucleotide changes (G₆₉ > A and A₂₅₁ > U). To further test the infectivity of the identified viroid, RNA extracts from the RG and VC graft-inoculated citrons were slash-inoculated onto four new citrons budded on rough lemon (C. jambhiri Lush.) rootstock. All citrons developed symptoms, and CVd-V was identified with RT-PCR, cloning, and sequencing. The CVd-V identification results were independently verified by CDFA’s Plant Pest Diagnostic Center, and the original source trees were destroyed. CVd-V was first reported in Spain (Serra et al. 2008a) and has been detected in various samples including a source from UCR, but the source of the UCR field material was not recorded (Serra et al. 2008b). This note reports the natural occurrence of CVd-V in CA and corroborates the CVd-VCᴬ variant report from the UCR source (Serra et al. 2008b). This note also demonstrates the importance of regulatory testing of propagative materials and elimination of infected citrus germplasm source trees to prevent the spread of viroids into commercial citrus.