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First report of three bunya-like viruses, apple luteovirus 1, and apple

hammerhead viroid in apples from Hakkari, Türkiye

Article in Plant Disease · December 2023

DOI: 10.1094/PDIS-10-23-2151-PDN

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Hakkari University USDA Animal and Plant Health Inspection Service
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First report of three bunya-like viruses, apple luteovirus 1, and apple

hammerhead viroid in apples from Hakkari, Türkiye

N. Akdura1, J. Mendoza2, S. Hasselhoff2, L. Costa2, X. Hu2, Y. Yang2, J.A. Foster2, C.

McFarland3, and O. P. Hurtado-Gonzales2†

1Department of Mathematics and Science Education, Faculty of Education, Hakkari University, Hakkari,
Türkiye
2 USDA-APHIS-PPQ, Plant Germplasm Quarantine Program, Beltsville, MD 20705, USA
3 USDA-APHIS-PPQ-Field Operations, Raleigh, NC, 27606, USA

† Correspondence: oscar.hurtado-gonzales@usda.gov (OPHG)

Türkiye is a major apple producer in the crossroads of Europe and the Middle East.
Several reports have described the presence of multiple viruses affecting apple
production in Türkiye, including apple stem grooving virus (ASGV), apple stem pitting
virus (ASPV), apple chlorotic leafspot virus (ACLSV), and apple mosaic virus (ApMV)
(Kurçman 1977; Fidan 1994; Çağlayan et al. 2003). However, there are no reports of the
presence of the recently discovered bunya-like viruses, namely citrus concave gum-
associated virus (CCGaV), and apple rubbery wood viruses 1 and 2 (ARWV1 and 2), as
well as apple luteovirus 1 (ALV-1), and apple hammerhead viroid (AHVd) in Türkiye, all
of which have been previously reported in other apple-producing countries (Wright et al.
2018; Liu et al. 2018; Zhang et al. 2014). Leaves from one ‘Gala’, two ‘Granny Smith’,
and one ‘Golden Delicious’ apple trees showing mild symptoms of curling, chlorosis, and
yellowing were collected from four different orchards in the province of Hakkari, southeast
Türkiye during June 2022 and sent to USDA APHIS Plant Germplasm Quarantine
Program (under permit P526P-21-02130) for virus and viroid high throughput sequencing
(HTS) diagnostics. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen)
following the manufacturer’s guidelines to prepare RNAseq libraries using the TruSeq
Stranded Total RNA Library Plant Kit (Illumina, Inc), as described in Malapi-Wight et al.
(2021). Libraries were sequenced on the NextSeq500 sequencer (pair end 2x75).
Approximately 45 million reads were obtained per sample on average. Bioinformatic
analysis was performed as described in Costa et al. (2022) using Phytopipe, where
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unclassified pathogen-derived reads were de novo assembled and contigs were

compared to the NCBI viral nucleotide and protein databases by BlastN and BlastX,
respectively, using a 10-4 e-value cutoff. Nearly complete genome contigs were obtained
for ACLSV (OR640150) and ASPV (OR640151) in all four samples and for ASGV
(OR640152) in three of the four samples (both Granny Smith trees and Golden Delicious).
The average BlastN identity to sequences in GenBank was 92.3% for ACLSV, ranging
from 89-94 %. BlastN identity for ASPV was 86%, ranging from 81-92 % while the ASGV
average BlastN identity was 98.2%. Nearly complete genomes with average genome
coverage of 92.4% and 95.6% for RNA1 and RNA2 of CCGaV (OR640153 and
OR640154), were found in two of the four samples (Gala and Golden Delicious) with
BlastN identity of 94.7% and 94.8%, respectively to GenBank sequences. Additionally, a
nearly complete sequence of the large (L), medium (M), and small (S) segments for
ARWV1 was found in two samples (both Granny Smith) with average genome coverage
of 99.9%, 99.4%, and 100%, respectively, and BlastN identity of 98.8%, 95.2%, and
98.4% (OR640155, OR640156, OR640157). ARWV2 contigs were also found in one
sample (Golden Delicious), where M and S segments had a coverage of 99.8% and
BlastN identity of 95.4% (OR640158 and OR640159). The nearly complete genome of
ALV-1 was also found in two of four samples (Granny Smith and Golden Delicious) with
genome coverage of 94.1% and an average BlastN identity of 93.4% (OR640160). AHVd
was found in one of the Granny Smith trees with 19,260 mapped reads to the reference
GenBank MH049335.1 and identity of 98.3% (OR640149). The HTS findings of ACLSV,
ASPV, ASGV, CCGaV, ARWV1, ARWV2, ALV-1, and AHVd from Türkiye were later
confirmed by Sanger sequencing using custom-designed primers targeting the coat
protein, the RNA-dependent RNA polymerase, or ~390bp for the AHVd genome
(Supplementary Table 1). To further learn about the incidence of CCGaV, ARWV1,
ARWV2, ALV-1, and AHVd, we tested 12 other apple samples from six different
neighboring orchards and found them at a 18.8% rate for CCGaV, 12.5% for both ARWV1
and ARWV2, 25% for ALV-1, and 37.5% for AHVd. To our knowledge, this is the first
report of CCGaV, ARWV1, ARWV2, ALV-1, and AHVd infecting apples in Türkiye. Further
studies of the impact of these agents on orchard’s health are necessary, including their
prevalence in high apple production regions of Türkiye.
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References

Çağlayan, K. et al. (2003) First report of molecular identification of apple viruses in Turkey.
J. Turk. Phytopath 32(2): 57-60.

Costa, L. et al. (2022) High-throughput detection of a large set of viruses and viroids of
pome and stone fruit trees by multiplex PCR-based amplicon sequencing. Front. Plant
Sci. 13: 1072768.

Fidan, Ü. (1994) Indexing of apple trees for apple mosaic virus, apple chlorotic leaf spot
virus and apple stem grooving virus by ELISA. J. Turk. Phytopath 23(3): 127–132.

Kurçman, S. (1977). Determination of virus diseases on cultural plant in Turkey. J. Turkish

Phytopath, 6(1): 25-46.

Liu, H. et al. (2018) Characterization of a new apple luteovirus identified by high-

throughput sequencing. Virology J. 15(2): 1-9.

Malapi-Wight, M. et al. (2021) HTS-based diagnostics of sugarcane viruses: seasonal

variation and its implications for accurate detection. Viruses 13(8): 1627.

Wright, A. A. et al. (2018) Diversity of three bunya-like viruses infecting apple. Arch. Virol.
163: 3339-3343.

Zhang, Z. et al. (2014) Discovery of replicating circular RNAs by RNA-seq and

computational algorithms. PLoS Path. 10.12: e1004553.
 Page 4 of 5

First Report of three Bunya-like viruses, apple luteovirus 1, and apple hammerhead viroid
in Apples from Hakkari, Türkiye

N. Akdura1, J. Mendoza2, S. Hasselhoff2, L. Costa2, X. Hu2, Y. Yang2, J.A. Foster2, C. McFarland3,

and O. P. Hurtado-Gonzales2†
1
Department of Mathematics and Science Education, Faculty of Education, Hakkari University, Hakkari, Türkiye
2
USDA-APHIS-PPQ, Plant Germplasm Quarantine Program, Beltsville, MD 20705, USA
3
USDA-APHIS-PPQ-Field Operations, Raleigh, NC, 27606, USA

† Correspondence: oscar.hurtado-gonzales@usda.gov (OPHG)

Supplementary Table 1.- Primers designed to confirm the presence of apple viruses and a viroid
by one-step reverse transcription polymerase chain reaction (RT-PCR) followed by Sanger
sequencing.

Virus/Viroid Targeted Amplicon

Oligo name Primer sequence (5’- 3’)
Name Region size (bp*)

CCGaV-PF CACTGCCAGTTAGCCAGTGA RdRp

Citrus concave 772
gum-associated CCGaV-PR TGCCGCAAAAGATGGTTTGG (RNA1)
virus
CCGaV-CF CTCTAGCAGTGGGTAGCAGC
Coat Protein 716
CCGaV-CR TCAGCTAGCAGAAATGTGGCA

ACLSV-PF TGCCATCAGCCCATCACTTT
Apple chlorotic RdRp 650
ACLSV-PR CCCTGCCTCCTCACGAAATT
leaf spot virus
ACLSV-CF GGAAAAGGAAAGGTTGGGACC
Coat Protein 666
ACLSV-CR ACAGACGAGAGCTTTGCCTC

ASPV- PF CTTGTCGACCTCATCCCAGG
RdRp 674
Apple stem pitting ASPV- PR ATGTCAGGGAGCATGTTCGT
virus ASPV-CF CAATCTCTGGCACCCATCGT
Coat Protein 747
ASPV-CR CCAGACATGCTTCGCGTAGA

Apple AHVd-F TCCGTTCCAAGGACGAAACC

hammerhead 395
viroid AHVd-R TGTTAAAACCCGTGCCCCAA
Page 5 of 5

ASGV-PF TGGGCCAACCACTCAGAAAG
RdRp 700
Apple stem ASGV-PR GGCTCAAGTACCACCCACAG
grooving virus ASGV-CF ATCTCTGCAAAAAGGGGGCC
Coat Protein 700
ASGV-CR TTCATCAGGCGTTAGACGGT

ALV1-PF GGTGTGGTTGTGGGAATTGC
RdRp 741
Apple luteovirus- ALV1-PR ACCTTGTGGCATAGCTGGTC
1 ALV1-CF GTATGGTCGTGCGTAGACGT
Coat Protein 607
ALV1-CP ACCTATTTGGGACCTTGCAA

ARW1-PF AAGTTTGGGACTGATGTCAGAT
RdRp 700
Apple rubbery ARW1-PR GAGTGGTCTGAGTCTCTGAGC
wood virus 1 ARW1-CF CTCTGATTTCTTGCAATGGCCA
Coat Protein 700
ARW1-CR ACAAGCCTAAATTGGACCATGC

ARW2-PF TCAAGATATGGAATCACTGAAAACATG
RdRp 720
Apple rubbery ARW2-PR GCTAGTTAGCTCACTTAAATCTAACTT
wood virus 2 ARW2-MPF GGAGACATTCCTTTTTCCGAAGA Movement
735
ARW2-MPR GGACTCTTCTGCAAAGCCCT Protein

*bp=base pair

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