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ORIGINAL RESEARCH
published: 10 July 2020
doi: 10.3389/fmicb.2020.01627

Edited by:
Ahmed Hadidi,
Agricultural Research Service (USDA),
United States
Reviewed by:Francesco Di Serio,
Istituto per la Protezione Sostenibile
delle Piante, Italy
Jeremy Thompson,
Cornell University, United States
Wilhelm Jelkmann,
Julius Kühn-Institut, Germany*Correspondence:
Nobuyuki Yoshikawa
yoshikawa@iwate-u.ac.jp
Specialty section:
This article was submitted to
Microbe and Virus Interactions with
Plants,a section of the journal
Frontiers in Microbiology
Received: 26 March 2020
Accepted: 22 June 2020
Published: 10 July 2020
Citation:
Li C, Yaegashi H, Kishigami R,
Kawakubo A, Yamagishi N, Ito T and
Yoshikawa N (2020) Apple Russet
Ring and Apple Green Crinkle
Diseases: Fulfillment of Koch’s
Postulates by Virome Analysis,
Amplification of Full-Length cDNAof Viral Genomes, in vitro
Transcription of Infectious Viral RNAs,and Reproduction of Symptoms on
Fruits of Apple Trees Inoculated With
Viral RNAs. Front. Microbiol. 11:1627.
doi: 10.3389/fmicb.2020.01627

Apple Russet Ring and Apple Green
Crinkle Diseases: Fulfillment of
Koch’s Postulates by Virome
Analysis, Amplification of Full-Length
cDNA of Viral Genomes, in vitro
Transcription of Infectious ViralRNAs, and Reproduction of
Symptoms on Fruits of Apple Trees
Inoculated With Viral RNAs
Chunjiang Li 1 , Hajime Yaegashi 2 , Ryusuke Kishigami 1 , Ayaka Kawakubo 1 ,Noriko Yamagishi 3 , Tsutae Ito 2 and Nobuyuki Yoshikawa 1,3*
Faculty of Agriculture, Iwate University, Morioka, Japan, 2 Division of Apple Research, Institute of Fruit Tree and Tea Science,
National Agriculture and Food Research Organization, Morioka, Japan, 3 Agri-Innovation Center, Iwate University, Morioka,
Japan
1

Apple russet ring and apple green crinkle are graft-transmitted diseases first reported
more than 60 years ago, but at present, no association between a specific virus (variant)and the disease has been clearly demonstrated. In this study, we conducted thefollowing series of experiments to identify the causal viruses (variants) of these apple
diseases; (1) comprehensive analysis by next-generation sequencing of all viruses ineach apple tree affected with russet ring or green crinkle disease, (2) amplification of
full-length genomic cDNA of viruses using primers containing the T3 promoter and thein vitro transcription of infectious viral RNAs, (3) inoculation of viral RNA transcripts toboth herbaceous and apple plants, (4) analysis of sequence variants of viruses present in
infected plants, (5) back-inoculation of sequence variants of candidate viruses to apple
seedlings combined with the virus-induced flowering technology using the apple latent
spherical virus vector to reproduce the symptom on the fruit as soon as possible, and(6) reproduction of symptoms on the fruits of apple trees inoculated with sequencevariants and the re-isolation of each virus variant from apples showing fruit symptoms.
The results showed that one of the sequence variants of the apple chlorotic leaf spot
virus causes a characteristic ring-shaped rust on the fruits of infected apple trees andthat a sequence variant of the apple stem pitting virus probably causes green crinkle

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symptoms on an infected apple fruit. Thus, we were able to fulfill Koch’s postulates toprove the viral etiology of both the apple russet ring and green crinkle diseases. We
also propose an experimental system that can prove whether a virus found in diseased
tissues is the pathogen responsible for the diseases when the etiology is undetermined.
Keywords: apple russet ring disease, apple green crinkle disease, next generation sequencing, apple chlorotic
leaf spot virus, apple stem pitting virus, Koch’ postulates

INTRODUCTION

to occur worldwide and implicated to be caused by a strain(or by strains) of ACLSV (van der Meer, 1986; Desvignes andBoye, 1988; Wood, 2001; Howell et al., 2011). However, manystrains or isolates of ACLSV are naturally in apple trees thatare both unaffected and affected by russet ring disease (Yaegashi
et al., 2007, 2011). In order to demonstrate that ACLSV may
be responsible for apple russet ring disease, it is necessary toisolate and characterize the strain and its sequence variants at themolecular level, and to back-inoculate these into apple plants for
symptom reproduction.
Apple green crinkle disease was first observed in 1929 in
Aomori Prefecture, Japan and the graft-transmission of thisdisease was demonstrated in Japan in 1934 (Sawamura, 1965),
thus this disease is the world’s first reported virus-like diseaseaffecting apple plants. The disease was also reported in Canada
and New Zealand in the 1930s (Chamberlain et al., 1974).
Symptoms occur only in the fruit and appear most clearly
during the young fruit stage, wherein some of the fruit are notenlarged and are irregularly dented, resulting in abnormal fruit
morphology. The pericarp of the indented part will later cork andbecome rust-like, and in severe cases, it will tear (Chamberlain
et al., 1974). To date, multiple viruses such as ASPV, ACLSV, and
ASGV etc. have been detected in trees affected by green crinkle
disease (Yaegashi et al., 2007; James et al., 2013). Among them,
ASPV has been implicated as the causal virus, but the relationshipbetween ASPV isolates and crinkle symptoms of fruits has notbeen demonstrated (Howell et al., 2011). James et al. (2013)
reported that a putative new foveavirus closely related to ASPV,
namely the apple green crinkle associated virus (AGCaV), hadbeen isolated from apples showing severe crinkle disease.
In this study, we conducted the following series of experiments
to determine the causal viruses (variants) of the apple russet
ring and green crinkle diseases: (1) comprehensive analysis
by next-generation sequencing (NGS) of all viruses in eachapple tree affected by russet ring or green crinkle diseases,(2) amplification of the full-length genomic DNA of viruses
using primers containing the T3 promoter and the in vitrotranscription of infectious viral RNAs, (3) inoculation ofvirus RNA transcripts to herbaceous and apple plants, (4)analysis of sequence variants of viruses in infected plants,(5) back-inoculation of sequence variants of candidate viruses
to apple seedlings combined with virus-induced flowering
technology using the apple latent spherical virus (ALSV)
vector (Yamagishi et al., 2014, 2016; Yamagishi and Yoshikawa,2016), and (6) reproduction of symptoms on the fruits ofapple trees inoculated with sequence variants and the reisolation of each virus variant from apples showing fruit
symptoms. From the results of the above studies, we were

There are many virus-like diseases affecting deciduous fruit trees
whose causal agents are not yet identified (Németh, 1986; Howell
et al., 2011). Even if a certain virus is presumed to be the causeof the disease, the causal relationship between the presence of avirus and specific symptoms has not been conclusively provenin many fruit tree viral diseases because back-inoculation of
the virus to the original fruit trees has not been achieved; that
is, Koch’s postulate has not yet been fulfilled. There are several
reasons why it is difficult to identify the causative virus of fruit
tree diseases. Firstly, many viruses are not so easy to isolate into
herbaceous test plants and/or to back-inoculate the isolated virus
into their original fruit trees. Secondly, some viruses of fruit trees
have not only difficulties to be transmitted to herbaceous hostplant, but they simply have a limited host range. This makes
it difficult to isolate the causative agent as a single virus. The
third reason is that the infection pattern of viruses is oftenvery complex in fruit trees. For example, reports show that asingle cherry tree affected by a bud blight disease was infectedwith 7 – 8 different virus species at the same time, and thatmost of these viruses could not be transmitted and isolated in
herbaceous test plants (Isogai et al., 2004; Yaegashi et al., 2020).
In Japan, most apple trees are infected with viruses such asapple chlorotic leaf spot virus (ACLSV), apple stem pitting virus(ASPV), and apple stem grooving virus (ASGV), etc. without
showing obvious symptoms (Yaegashi et al., 2011). In addition,
each virus (ACLSV, ASGV, or ASPV) in single apple trees consistsof a mixture of sequence variants that differ considerably fromone another in terms of nucleotide sequences (Candresse et al.,
1995; Yoshikawa et al., 1996, 2001; Magome et al., 1997b, 1999;
Yaegashi et al., 2007). The complexity of the virus infection
patterns in fruit trees makes it difficult to identify the virus
or virus variant responsible for a specific disease. Examples ofgraft-transmitted diseases of apples with undetermined etiology
include the apple russet ring and apple green crinkle diseases(Howell et al., 2011).
The apple russet ring is a graft-transmitted disease of apple inwhich a characteristic ring-shaped rust appears on the fruits. The
disease was firstly reported on a ‘McIntosh’ apple that showed leaf
pucker symptoms in 1954 in British Columbia, Canada (Wood,1971). The russet ring symptom of the fruit was shown to be
associated with leaf pucker (Wood, 1971). The graft transmissionof the russet ring disease was reported on the ‘Golden Delicious’apple from Washington state, United States (Wood, 1971); inJapan, the russet ring disease was found in the ‘Mutsu’ apple
from Iwate Prefecture in 1972 and was also shown to be graftedtransmitted (Yanase et al., 1988). The disease is now reported

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able to prove the specific sequence variants of the causal
viruses of the two apple diseases. Our study establishes anexperimental system to identify the causal virus of fruit
tree diseases with undetermined etiology by isolation of theviruses using the amplification of full-length cDNA of viralgenomes followed by in vitro transcription of infectious viralRNAs and reproduction of symptoms on original hosts byback inoculation.

800 µL of RNA extraction buffer [2% CTAB, 2% PVP K30, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0), 2
M NaCl, 2% mercaptoethanol]. Samples in microtubes werecrushed using a Micro Smash MS-100R (TOMY SEIKO) andincubated at 65 C for 20 min with occasional stirring. After the
addition of 800 µL of chloroform, the extracts were sufficiently
stirred and centrifuged at 10,000 ⇥ g for 10 min (4 C). Then,
700 µL of the supernatants were added to 233 µL of 7.5
M LiCl, sufficiently mixed, and left at 4 C overnight. Aftercentrifugation at 14,000 ⇥ g for 25 min, the pellets were added
to 1 mL of 80% ethanol and centrifuged at 14,000 ⇥ g for
5 min. The pellets were then suspended in sterilized distilledwater (SDW) to obtain the total RNA solutions. Total RNA
concentrations were measured by NanoDrop (Thermo Fisher
Scientific, Tokyo, Japan).
The coat protein (CP) region of each virus was amplified
through RT-PCR as follows: 1 µL of total RNA solution(1 µg/µL), 0.5 µL of Oligo (dT)12 primer (10 µM), 4 µL
of 2.5 mM dNTP mixture (TaKaRa Bio Inc., Kusatsu, Japan),
2 µL of 5 ⇥ RT Buffer (TOYOBO Co., Ltd., Tokyo, Japan),
0.5 µL of ReverTra Ace (100 unit/µl) (TOYOBO), and 2 µLof SDW were mixed and incubated at 42 C for 60 min using
the TaKaRa PCR Thermal Cycler Dice Version II (TaKaRa)
for reverse transcription reaction. PCR was then conductedusing TaKaRa Ex Taq and the following primer pairs: SPCP8148-8167 (+) [50 -ttcgaccctaaccttcatgg-30 ] and SP-CP88708890 (–) [50 -ctttgagtttgcagcatgagg-30 ], with the expected productcorresponding to base numbers 8148 to 8890 of ASPV-IF38(accession no. AB045371) (Yoshikawa et al., 2001) for the
ASPV-CP region, AC-CP 6821-6840 (+) [50 -agatctgaaagcgttcctg30 ] and AC-CP7342-7365 (–), with the expected product iscorresponding to bases 6821–7365 of ACLSV-P205 (accession no.
D14996) (Sato et al., 1993) for the ACLSV-CP region, and SGCP5590-5611(+) (50 -aaagagaaagtttaggtccctc-30 ) and SG-CP63956413 (–) (50 -taaaggcaggcatgtcaac-30 ) corresponding to bases
5590–6413 of ASGV-P209 (accession no. D14995) (Yoshikawa
et al., 1992). The PCR conditions were: 35 cycles of 94 C for
30 s, 58 C for ASPV, 54 C for ACLSV, and 55 C for ASGV for
30 s, 72 C for 1 min. PCR products were electrophoresed on
a 1% agarose gel, purified using MonoFas DNA purification
kit (GL sciences), and then TA cloning was performed using
the TaKaRa Mighty TA-cloning Kit (TaKaRa). Plasmids wereextracted from Escherichia coli and used for sequence analysis
as descried below.

MATERIALS AND METHODS
Plants and dsRNA Extraction
An apple tree (PK-51, ‘Royal Gala’) affected with the apple
russet ring disease (ARRD) and an apple tree (P-190, ‘Golden
Delicious’) with the apple green crinkle disease (AGCD)
were maintained in the field of the Division of Apple
Research, Institute of Fruit Tree and Tea Science, NARO in
Morioka, Iwate prefecture, Japan. These apple trees show typicalsymptoms of russet-ring and green crinkle on fruits, respectively(Supplementary Figure S1). dsRNAs were extracted from the
newly developed leaves of an apple tree PK-51 infected with
ARRD and an apple tree P-190 infected with AGCD. Leaf
samples (20 g) were collected from diseased apple trees andstored at 80 C until use. Extraction of dsRNA from the leavesof diseased trees were performed using cellulose powder as
described previously (Morris and Dodds, 1979; Yoshikawa andConverse, 1990), and the dsRNA was dissolved in 20 µL TAE
buffer (40 mM Tris, 20 mM sodium acetate, 1 mM EDTA, pH
7.0). The dsRNA was electrophoresed on a 5% polyacrylamide
gel and stained with silver nitrate to determine the presence ofdsRNAs (Yoshikawa and Converse, 1990).

R

Next-Generation Sequencing andComputational Analysis
dsRNAs from the ARRD or AGCD-infected samples were thensubjected to library preparation as reported previously (Nodaet al., 2017; Yaegashi et al., 2020). The prepared library was
analyzed using 6% polyacrylamide gel electrophoresis and the
products (200–300 bp) were purified and subjected to pairedend sequencing (read length 101 bases) using the HiSeq2000(Illumina Inc., San Diego, CA, United States). Resulting reads
were assembled de novo into contiguous sequences (contigs)
using Velvet (ver. 1.2.08) (Zerbino and Birney, 2008) with k = 97
in RR and k = 95 in GC samples. The obtained contigs (>500 bp)
were then used to search for virus sequences using the blast
program of NCBI BLAST+.1

R

In vitro Transcription of Virus Genomic
RNA
Synthesis of full-length DNA from the genomic RNAs of
ACLSV, ASPV, and ASGV was done by subjecting total
RNA samples to a reverse transcription reaction at 42 C
for 60 min using the PrimeScript II RT enzyme andoligo dT primer from the PrimeScript High Fidelity RTPCR Kit (TaKaRa) according to manufacturer protocols.
Full-length DNAs of the three viruses were amplified byPrimeSTAR GXL DNA polymerase (TaKaRa) using primer pairs
containing the T3 promoter sequence: ACLSV5’endT3(+) (50 aattaaccctcactaaagtgatactgatacagtgtacact-30 ) and ACLSV3’end(–)

RT-PCR
Leaves and petals were collected from apple trees affected by
ARRD (PK-51) and AGCD (P-190) and were stored frozen at
80 C. Frozen samples (1 g) of each affected tree were put
into a 2.0 mL microtube together with stainless steel beads
SUB-50 (TOMY SEIKO Co., Ltd., Tokyo, Japan) and added1

R

https://blast.ncbi.nlm.nih.gov/Blast.cgi

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Apple RR and GC Diseases(50 -tttttttagtagtaaaatatttaaaagt-30 ) for ACLSV, ASPV50 endT3(+)(50 -aattaaccctcactaaaggatacgcaaacaaactctg-30 and ASPV30 end( )(50 -ttttttgaaaatctagttaaaacaaaaataag-30 ) for ASPV, and ASGV50
endT3(+) (50 -aattaaccctcactaaaggaaatttaacaggcttaattt-30 ) and
ASGV30 end( ) (50 -ttttttttttttttttttttagagtggacaaactctagac-30 ) for
ASGV. PCR was performed as follows: 98 C for 30 s, 54 C for
ACLSV, 59 C for ASPV, and 52 C for ASGV for 30 s, and 68 C
for 10 min for 40 cycles. The PCR product was then purified
using the MonoFas DNA Purification Kit II (GL Sciences
Inc., Tokyo, Japan). Purified full-length genomic DNAs werethen used as templates for the in vitro transcription of fulllength genomic RNA of each virus using the MEGAscript Kit(Ambion, Thermo Fisher scientific, Tokyo, Japan). The reaction
mixtures contained the full-length genomic cDNA of each virus,
ATP, CTP, UTP (75 mM each), GTP (15 mM), Cap Analog(40 mM) Enzyme Mix, and RNasin Plus RNase Inhibitor(Promega, Madison, United States), and were incubated for3.5 h at 37 C. The reaction mixtures were then treated with
TURBO DNase (Thermo Fisher scientific) (2 U/µl) at 37 C for
15 min to decompose the template DNAs. RNA samples werethen extracted using water-saturated phenol and chloroform,
precipitated using ethanol, and suspended in SDW. RNAtranscript concentrations were measured using NanoDrop, while
RNA transcript quality was also checked using 1% agarose gelelectrophoresis.

using the ALSV vector (Yamagishi et al., 2014, 2016; Yamagishi
and Yoshikawa, 2016) for symptom reproduction on the apple
fruits. VIF using the ALSV vector can reduce flowering 2 months
after germination and lead to fruit formation within 1 year inapple seedlings (Yamagishi et al., 2014).
Total RNA samples extracted from infected N. occidentalis
leaves were used as templates for generating full-length cDNAof each sequence variant of both ACLSV and ASPV, followed
by in vitro transcription of infectious viral RNA, as describedabove. As inoculum sources for inoculation to apple seedlings,
we selected five N. occidentalis plants infected with RRACV1,RRACV2, RRACV3, RRACV5, or RRACV1 + RRACV4. In thecase of ASPV variants, the RNA transcripts of ASPV fromthe AGCD-sample were also inoculated to apple seedlings with
the ALSV-AtFT/MdTFL1 vector through particle bombardment,
as described above.
Immediately after germination, cotyledons of apple seedlings
from seeds of ‘Ourin’ were biolistically inoculated with the
RNA transcript of each viral RNA, along with the RNAof ALSV-AtFT/MdTFL1 as described previously (Yamagishi
et al., 2010, 2016). Inoculation was performed in a heliumpressure of 1379 kilopascal (kPa) using the Helios Gene Gunsystem (Bio-Rad Laboratories, München, Germany), with two
shots used per cotyledon (4 shots in total). Inoculated apple
seedlings were incubated in a growth chamber at 25 C for
ACLSV and at 20 C for ASPV, with the day length set to
16 h for approximately 2 months, and then grown in a noncontaminant greenhouse.
Infection assay was conducted by RT-PCR as described
before (Yamagishi et al., 2014) using primer pairs for
ACLSV [ACCP6821-6840(+) (50 -agatctgaaagcattcctg-30 ) and
ACCP7342-7365( ) (50 -ctaaatgcaaagatcagttgtaac-30 )], ASPV[SPCP8148-8167(+) (50 -ttcgaccctaaccttcatgg-30 ) and SPCP8
870-8890( ) (50 -ctttgagtttgcagcatgagg-30 )], ALSV-RNA1 [ALSVRNA16598(+) (50 -gtacattcctcccaatcaaag-30 ) and ALSVRNA
16691(–) (50 -ggatcaggagaacaaactag-30 )], and ALSV-RNA2 [ALS
VRNA21418(+) (50 -cccaaatctgctagaaggtc-30 ) and ALSVR
NA21511(–) (50 -gcaaggtggtcgtga-30 )]. When the infected apple
seedlings flowered, they were pollinated to produce fruits.

R

R

R

Inoculation of RNA Transcripts to Plants
and Sequence Analysis of Virus Variants
in Infected Plants
Inoculation of the RNA transcripts of ACLSV, ASPV, and ASGV
to Nicotiana occidentalis was conducted as follows: two leavesof N. occidentalis plants (6th to 7th leaf stage) were inoculatedwith 10 µL of viral RNA transcripts (0.5–1 µg/µl) per leaf,
along with carborundum. After inoculation, plants were grownin a growth chamber at 25 C with the day length set to
8 h. Chenopodium quinoa was also used for inoculation of the
ACLSV-RNA transcript.
Total RNA was extracted from the leaves of infected plants andthe CP region of each virus was amplified as described above. For
sequence analysis of virus variants in each infected plant, the PCR
products were purified using MonoFas DNA purification kit(GL sciences) and TA cloning was performed using the TaKaRa
Mighty TA-cloning Kit (TaKaRa). DNA plasmids were purified
using a Plasmid DNA Extraction Mini Kit (FAVORGEN Biotech,Ping-Tung, Taiwan) according to the manufacturer’s protocol.
The sequences of the cDNA clones were analyzed using a DNA
sequencing service from Sigma-Aldrich Japan. The homology of
the CP region sequence between DNA clones was analyzed using
the DNASIS software (Hitachi, Tokyo, Japan).

GraftingR

To confirm that RRACV2 induced ARRD in apple
seedlings as described above, graft-transmission tests
were conducted using apple seedlings infected with each
RRACV and ALSV-AtFT/MdTFL1. Approximately 1 year
after inoculation by particle bombardment inoculation,
the shoots of infected apple seedlings infected with eachsequence variant (SV) of ACLSV were cut and graftedto the stem of 3-year-old virus-free apple trees (‘Golden
Delicious’/JM7) using the side-grafting method for the
reproduction of russet ring symptoms on the fruits. Grafted
apple trees were named APT1 (an apple tree grafted withRRACV1), APT2 (with RRACV2), APT3 (with RRACV3),
APT1 + 4 (with RRACV1 + RRACV4), and APT5 (withRRACV5). Inoculated apple plants were grown in agreenhouse for 4 years at the Apple Research Center, NARO
in Morioka, Japan.

Back-Inoculation of Apple Seedlings
With ACLSV and ASPV
Both ARRD and AGCD cause characteristic symptoms on apple
fruits as shown in Supplementary Figure S1. However, it
generally takes at least 6–7 years for apple seedlings to bloom.
For this reason, we used virus-induced flowering (VIF) system

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Nucleotide Sequencing of the Complete
Genome of ACLSV and ASPV Variantsof ACLSV infection (Supplementary Figure S4). N. occidentalis
plants infected with ASGV did not show any obvious symptoms.
RT-PCR using a primer pair for each virus showed that only
ASPV was detected in plants inoculated with the ASPV RNA
transcript, and the other two viruses (ACLSV and ASGV)
were not detected (Supplementary Figure S3). Similarly, only
ACLSV was detected in plants inoculated with the ACLSV RNA
transcript (Supplementary Figure S3), and only ASGV wasdetected in plants inoculated with the ASGV-RNA transcript(Supplementary Figure S3). Thus, by the inoculation of in vitrotranscripts of viral RNAs, each of the three viruses co-infecting anapple tree could be isolated into separate plants.

DNA fragments covering entire genomes of the sequencevariants of both ACLSV and ASPV were amplified from samples
infected with each variant using the primer pairs shown in
Supplementary Tables S1, S2. TA cloning of the amplified
sequences was conducted as described above. Nucleotide
sequencing of the cDNA clones was performed using a DNA
sequencing service (Sigma-Aldrich, Japan).

RESULTS
Virome Analysis of Apple Trees Affectedby ARRD and AGCD

Complexity of Sequence Variants of
ACLSV in an Apple Tree (PK-51) Affectedby ARRD

Using NGS analysis of dsRNAs (Supplementary Figure S2)from the ARRD and AGCD samples in this study, we obtained
51,514,584 reads from the ARRD sample and 70,424,624 reads
from the AGCD sample (Table 1). The conventional de novo
assembly of the reads from ARRD and AGCD samples generated
411 contigs with an average of 2,095 base pairs (bp) and 853
contigs with an average of 771 bp, respectively (Table 1). In a
subsequent blastn analysis, 63 contigs from the ARRD-sample
and 244 contigs from the ACGD-sample corresponded to virus
sequences (Table 1). In the ARRD sample, ACLSV had thelargest number of contigs (42.9% of the total), followed by ASPV
(13%), ASGV (6.4%), AGCaV (3.2%), and apricot latent virus(ApLV) (1.6%) (Table 1). On the other hand, in the GC sample,
ASPV was the largest, accounting for 34.4% of the total, followed
by ASGV (8.2%), ACLSV (7.8%), AGCaV (2.9%), and ApLV
(2.9%) (Table 1).

We analyzed the nucleotide sequence of the selected 18 clonesof the ACLSV-CP region amplified from the ARRD-infected
apple tree (PK-51). Here, those with nucleotide sequence identityof 99% or more in the CP region (582 nt) were regarded
as deriving from the same SV. The results indicate that atleast six sequence variants (SV1⇠SV6) were present in samples
from an apple with ARRD (Supplementary Figure S5). The
detection frequency of each SV was different and the detected
numbers of each SV in the total 18 clones analyzed were
5 clones for SV1, 2 clones for SV2, 1 clone for SV3, 4
clones for SV4, 6 clones for SV5, and 1 clone for SV6.
Sequence identities of the CP region between SVs of ACLSV
were 83.3–95.1% in terms of nucleotides and 91.7–97.9% interms of amino acids.
In subsequent experiments, we analyzed the SVs in 17
herbaceous plants (N. occidentalis and C quinoa) infectedwith the RNA transcript of the ACLSV genome. Nucleotide
sequence analysis of DNA clones from 17 infected plants (10clones/plant) indicated that five SVs (named RRACVs1 ⇠ 5)
were detected in infected plants (Figure 1). Among the 17
infected plants, most N. occidentalis plants were singly infectedwith either RRACV1 (11 plants), RRACV2 (2 plants), RRACV3(1 plant), or RRACV5 (2 plants), except for a plant infectedwith RRACV4 that was found to be co-infected with RRACV1.
Comparison of the RRACV1–RRACV5 nucleotide sequenceswith those of SV1-SV6 from an RR-apple in Supplementary
Figure S5, RRACV1 was the same as SV1, RRACV2 was SV2,RRACV3 was SV3, and RRACV4 was SV4. On the other hand,
RRACV5 was different from both SV5 and SV6 in an apple
with ARRD, suggesting that at least seven SVs were co-infecting
the RR-apple (PK-51). We could not find N. occidentalis plants
infected with SV5 and/or SV6 in this experiment. Sequence
identities of the CP region among RRACVs were 83.8–92.4%
in terms of nucleotides and 91.7–97.9% in terms of aminoacids (Table 2). The sequence identities between RRACVs 15, SVs 5 and 6, and isolates reported previously (P205 andB6) are shown in Table 2. It has been reported that ACLSV
can be divided into two types (P205 and B6) based on the CP
amino acid sequence (Yaegashi et al., 2007). Among the sevenSVs found in an ARRD-apple and inoculated N. occidentalis,

Isolation of Viruses Into Separate Plants
by Inoculation of in vitro Transcripts ofViral RNAs
Virome analysis by NGS revealed that both PK-51 and P-190
trees were infected with multiple viruses as described above.
In order to isolate each virus from a diseased apple trees into
separate herbaceous plants, the full-length DNA of the genomic
RNA of each virus was amplified from the total RNA of an apple
sample with ARRD (PK-51) using RT-PCR (Supplementary
Figure S3), and the obtained full-length viral RNAs weretranscribed in vitro using the full-length DNAs as templates. It
can be seen that RNAs corresponding to the size of each virus
genome [ASPV, 9.3 kb: ACLSV, 7.4 kb; ASGV, 6.4 kb] weresynthesized (Supplementary Figure S3). Each RNA transcript
was inoculated into N. occidentalis plants, and the infection was
assayed using RT-PCR, from which it was found that infection
was present in 17 plants of the 24 plants inoculated with the
ACLSV RNA transcript (infection rate, 71%), 5 out of 10 plants
inoculated with ASPV (50%), and 6 out of 6 plants inoculatedwith ASGV (100%). Infected N. occidentalis plants showed
symptoms consisting of mosaic and malformation of leaves(ACLSV) and of interveinal chlorosis (ASPV) (Supplementary
Figure S4). C. quinoa plants infected with ACLSV showedchlorotic spots and mosaic in the upper leaves, which is typical

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TABLE 1 | Summary of the conventional de novo assembly strategy of the reads from russet ring (RR)- and green crinkle (GC)-diseased apple samples.
Apple trees

No. of reads

Total no. of contigs
Total (mean, bp)

With virus hit*

No. of contigs with hit to virus**
With no hit

ACLSV

ASGV

ASPV

AGCaV

ApLV

othersPK-51 with RR

51,514,584

441 (2,095)

63

378

27

8

11

2

1

14

P-190 with GC

70,424,624

853 (771)

244

609

19

20

84

7

7

107

*No. of contigs with hit to viruses with significant E-value (<0.001). **ACLSV, apple chlorotic leaf spot virus; ASGV, apple stem grooving virus; ASPV, apple stem pitting
virus; AGCaV, apple green crinkle-associated virus; ApLV, apricot latent virus; others, stealth virus 1 etc.

FIGURE 1 | Alignment of the nucleotide sequences of CP region of ACLSV-RRACVs detected in leaf samples of N. occidentalis plants inoculated with the RNAtranscript of ACLSV from ARRD. Identical bases among the five SVs are filled in yellow, and if there are substitutions in the bases, they are filled in green (the number
is dominant) or unfilled (the number is inferior).

three SVs (RRACV3, RRACV4, and RRACV5) belong to theP205 type and four (RRACV1, RRACV2, SV5, and SV6)
belong to the B6 type.

RRACV1 + 4 (10–20 seedlings per each RRACV), the upper
leaves of apple seedlings were sampled, and virus infection
was examined by RT-PCR. Both ACLSV and ALSV weredetected in most of the inoculated apple seedlings (example
shown in Supplementary Figure S6). However, some seedlings
in which the AtFT gene was deleted from the ALSV vector
were observed as shown in Supplementary Figure S6. As
a result, 10 seedlings infected with RRACV1, 6 seedlings
with RRACV2, 4 seedlings with RRACV3, and 4 seedlings
with RRACV5 were used for further experiments. Infected

ACLSV-RRACV2 Induces Russet-Ring
Symptoms on Fruits of Infected Apple
Seedlings
Approximately 1 month after inoculation with viral RNA
transcripts of RRACV1, RRACV2, RRACV3, RRACV5, andFrontiers in Microbiology | www.frontiersin.org

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TABLE 2 | Identities of the nucleotide and amino acid sequences of the coat protein gene between the sequence variants (SV) and the isolates of ACLSV found in
infected N. occidentalis and an apple tree (PK-51) with ARRD.
SVs and isolates

SVs and isolates
RRACV1RRACV1RRACV291.0

RRACV3RRACV4RRACV5

SV5

SV6

P205

B6

85.4

83.8

85.0

95.1

93.4

84.5

91.2

87.1

86.0

85.5

91.7

89.1

85.2

96.0

91.7

92.4

86.4

85.7

89.7

86.6

91.7

84.8

83.3

87.1

86.4

84.7

82.8

88.4

86.2

92.7

83.7

90.5

83.6

88.3

RRACV297.9

RRACV393.2

92.7

RRACV4

92.2

91.7

97.4

RRACV5

97.9

91.7

97.9

92.2

SV5

97.9

96.8

92.7

91.7

91.7

SV6

92.2

97.4

91.7

97.9

90.6

96.3

P205

91.7

91.7

96.3

94.3

96.4

90.7

90.1

B6

97.9

97.9

94.3

92.7

92.7

97.4

96.4

85.0
90.2

The percentages of nucleotide sequence (582 bases) identities are above the diagonal and the percentages of amino acid sequence (193 aa) identities are below thediagonal. RRACV1⇠5 was found in infected N. occidentalis, and both SV5 and SV6 was found in an ARRD-apple. P205 is an isolate from apple (Sato et al., 1993) andB6 is an isolate from an ARRD-apple (Yaegashi et al., 2007).

seedlings showed early flowering from approximately 2–
3 months after inoculation, and the flowered plants wereartificially pollinated. Finally, the number of apple seedlings
with fruits was found to be one with RRACV1, two withRRACV2, two with RRACV3s, and one with RRACV5.
We could not obtain apple seedlings with fruits infectedwith RRACV1 + 4.
Among the infected apple seedlings, a plant inoculatedwith RRACV2 (No. 1) showed a ring and line patternof chlorosis on the leaf about 2 months after inoculation(Figure 2A, left). The fruits of the pollinated seedlings grew
to 5–6 cm in diameter 5 months after pollination. In plant
No. 1 infected with RRACV2, a dark green ring was observedon the fruits as shown in Figure 2A, center. Ring-shaped
rust was observed on the fruit of another seedling infectedwith RRACV2 (no. 2) (Figure 2A, right). Though theseapple seedlings were co-infected with ACLSV-RRACV2 and
ALSV-AtFT/MdTFL1, ALSV-AtFT/MdTFL1 has never caused
any symptoms in both leaves and fruits of infected apple
seedlings (Yamagishi and Yoshikawa, 2016), and it is most
likely that the symptoms observed were induced by RRACV2
infection. No symptoms were observed on either leaves orfruits of apple seedlings infected with RRACV1, RRACV3, or
RRACV5. RT-PCR assays indicated that ACLSV was detectedin fruit peel and flesh tissues of apple seedlings infectedwith RRACV1, RRACV2, RRACV3, or RRACV5, regardless ofwhether symptoms were observed.

of chlorosis on leaves as shown in Figure 2B; however, no
symptoms were found on the fruits obtained this year. Three
years after grafting, russet-ring symptoms typically of ARRD
were observed on the fruits of APT2, in addition to ringand line-patterns on leaves (Figure 2B). Other apple trees(APT1, APT3, APT1 + 4, APT5, and non-grafted control
apple plants) did not show any symptoms on either leaves
or fruits even 4 years after grafting. Thus, only apple trees
inoculated with RRACV2 showed russet ring symptoms
typical of ARRD. Amplification of the CP regions by RT-PCR
and sequence analysis of the DNA products (6 clones perapple plant) from APT1, APT2, APT3, APT1 + 4, and APT5
indicated that only RRACV1 was detected from APT1, only
RRACV2 from APT2, only RRACV3 from APT3, and only
RRACV5 in APT5 (Supplementary Table S3). On the other
hand, only RRACV1 was amplified from APT1 + 4, suggestingthat RRACV4 might be lost from APT1 + 4 (Supplementary
Table S3). ALSV-AtFT/MdTFL1 was not detected in anyof the leaves of all apple trees (APT1–APT5) because wefound that it is very difficult to transmit ALSV from infected
to uninfected apple trees by grafting (unpublished result).
From these results, we concluded that ACLSV-RRACV2 is acausal agent of ARRD.
We have determined the complete nucleotide sequencesof the RRACV2 and RRACV1 genomes. The genome of
RRACV2 consists of 7557 nt excluding the 30 -polyA tail
(GenBank/EMBL/DDBJ accession LC533838) and encodes
three proteins from the 50 -terminus: an RNA replicationassociated protein (Rep) [216 kilo dalton (kDa), 1883 aa],a movement protein (MP) (50 kDa, 460 aa), and a CP
(21 kDa, 193 aa). The genome of RRACV1 consists of
7562 nt (accession LC533837) and contains three ORFs
encoding a Rep (216 kDa, 1885 aa), an MP (50 kDa, 460aa), and a CP (21 kDa, 193 aa). Phylogenetic analysis
based on the complete nucleotide sequence of the ACLSV
isolates showed that RRACV2 is grouped into the B6 type
and RRACV1 into the P205 type (Figure 3). The RRACV2
genome sequence was closest to that of B4 isolated fromACLSV-RRACV2 Causes ARRD in Apple‘Golden Delicious’
All grafted apple trees (APT1 with RRACV1, APT2
with RRACV2, APT3 with RRACV3, APT1 + 4 withRRACV1 + RRACV4, and APT5 with RRACV5) produced
fruits from a year after grafting, but no symptoms wereobserved on either leaves or fruits. Two years after grafting,the apple tree inoculated with RRACV2 (APT2) showedvirus-like symptoms consisting of ring and line patterns

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FIGURE 2 | Symptoms of the apple plants inoculated with ACLSV-RRACV 2. (A) Ring pattern of chlorosis on a leaf (left) and ring pattern on the fruits (center andright) of apple seedlings co-inoculated with RRACV2 and ALSV-AtFT/MdTFL1. (B) An apple tree (‘Golden delicious’) 3 years after grafting with cut branches infectedwith ACLSV-SV. Arrows in a left photo indicated the positions of grafting. Ring and line patterns of chlorosis on leaves of an apple tree (AP2) infected with
ACLSV-RRACV2 (upper right) and young fruits showing russet-ring symptom on an apple tree (AP2) infected with RRACV2 three years after grafting (lower right).
Scale bar; ca. 1 cm.

another apple tree affected by ARRD, and the identities ofnucleotide and amino acid sequences between RRACV2 andB6 were 89.4% (complete genome), 94.3% (Rep), 93.9% (MP),and 98.0% (CP).

multiple SVs, judging from the fluorescence peaks in thesequences of the amplified DNA (data not shown). Next,
the amplified DNAs from 5 infected N. occidentalis plants
were cloned as described above, and the resulting DNA
clones were sequenced (7–10 DNA clones per infected plant).
Among apple seedlings infected with both the ASPV-SV and
ALSV-AtFT/MdTFL1 vector, we selected five seedlings forthe amplification of their CP region, followed by cloning of
the DNA products; DNA clones (13–18 clones per infected
apple seedling) were also sequenced. Sequence analysis of
DNA clones from 10 infected plants (5 N. occidentalis and 5
apple seedlings each) indicated that a total of eight SVs weredetected in plants inoculated with RNA transcripts from apple(P-190) affected by AGCD. The SVs were named GCSPV1
to GCSPV8 (Supplementary Figure S8). Sequence identitiesof the amplified CP region of the ASPV genome (705 bp
excluding primer sequences) between GCSPV1 and GCSPV8

Eight ASPV-SVs Were Detected in an
Apple Tree (P-190) Affected by AGCD
RT-PCR analysis of inoculated N. occidentalis plants showedthat all plants (10 out of 10 inoculated plants) were infectedwith ASPV (Supplementary Figure S7). The PCR products(745 bp of the CP region of the ASPV genome) were purified
from 10 infected plants and analyzed by direct sequencing
using the SPCP8148-8167(+) and SPCP8870-8890( ) primer
pair. We expected that some plants were infected with asingle different SV, as was the case with RRACV; however,
all N. occidentalis plants were found to be infected with

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FIGURE 3 | Phylogenetic tree based on the complete nucleotide sequences of ACLSV genomes. The tree was generated by CLUSTALW program (DNA databank ofJAPAN [DDBJ]; URL: http://clustalw.ddbj.nig.ac.jp/). Bootstrap values for each node are shown (1000 replicates). Sequence variants analyzed in this study are
shown in red. Apple (RR*) indicated an apple tree affected with ARRD used in this study and apple (RR**) is an another apple tree affected with ARRD described
before (Yaegashi et al., 2007).

infected N. occidentalis plants and not from the infected
apple seedlings.

were 75.4–89.7% in terms of nucleotides and 83.8–91.8% interms of amino acid identities (Table 3). In total, 119 DNA
clones were sequenced, and the number of each SV clone
was observed to be as follows: 2 (1.7% of the whole) for
GCSPV1, 31 (26.1%) for GCSPV2, 10 (8.4%) for GCSPV3,2 (1.7%) for GCSPV4, 45 (37.8%) for GCSPV5, 12 (10.1%)
for GCSPV6, and 14 (11.8%) for GCSPV7, and 3 (2.5%) for
GCSPV8. The types and clone numbers of the SVs weredifferent in each infected plant. For example, the type (thenumber of clones) of GCSPV detected in apple seedlings
No. 1–No. 5 were GCSPV2 (13 clones) and GCSPV6 (1clone) in the No. 1 plant, GCAPV6 (9) and GCAPV7 (9)in the No. 2 plant, GCAPV5 (6), GCAPV6 (1), GCAPV7(3), and GCAPV8 (3) in the No. 3 plant, GCAPV5 (14)in the No. 4 plant, and GCAPV5 (12), GCAPV6 (1), and
GCAPV7 (2) in the No. 5 plant (Table 4). Three SVs(GCSPV1, GCSPV3, and GCSPV4) were detected only from

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ASPV-GCSPV2 Probably Induces Green
Crinkle Symptoms in the Fruit of
Inoculated Apple SeedlingApple seedlings (No. 1 and No. 5) inoculated with RNAtranscripts of ASPV and ALSV-AtFT/MdTFL1 vector flowered
2–3 months after inoculation and produced fruits, as shownin Figure 4. The infected apple seedlings did not show any
symptoms on leaves, but a fruit on the No. 1 apple seedlinginfected with GCSPV2 + GCSPV6 showed cracks on the
fruit surface in a manner similar to the symptoms of AGCD
(Supplementary Figure S1). Other apple seedlings (Nos. 2, 3,
4, and 5) infected with ASPV-SVs and control seedlings with
ALSV-AtFT/MdTFL1 only produced normal shaped apple fruits

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TABLE 3 | Identities of the nucleotide and amino acid sequences of the coat protein region (705 bp) between the sequence variants (SV) of ASPV found in
N. occidentalis and apple seedlings inoculated with the RNA transcript of ASPV from AGCD-apple.
GCSPV1GCSPV1GCSPV2GCSPV3GCSPV4GCSPV5GCSPV6GCSPV7GCSPV8

79.2

89.7

78.2

79.6

83.1

80.9

79.3

77.9

82.8

85.8

75.4

78.6

84.1

79.6

80.2

85.5

80.2

80.2

83.3

76.9

79.6

88.4

78.2

80.8

83.5

82.1

78.5

GCSPV287.2

GCSPV391.8

85.4

GCSPV4

86.6

89.3

85.5

GCSPV5

89.7

91.8

88.4

89.3

GCSPV6

88.8

85.5

89.7

84.6

88.0

GCSPV7

87.1

86.7

84.1

83.8

89.3

86.7

GCSPV8

89.3

90.1

87.6

90.6

91.0

86.8

80.5
86.7

TABLE 4 | Analysis of the sequence variants (GCSPVs) of ASPV found in leaves of apple seedlings (No. 1⇠ No. 5) inoculated with ASPV-RNA transcripts and
ALSV-AtFT/MdTFL1.
Plant no. apple seedlings

GCSPV (number of clone)

No. of clones sequenced

1

2

3

4

5

1

–

+ (13)

–

–

–

+(1)

2

–

–

–

–

–

3

–

–

–

–

4

–

–

–

–

0

13

0

0

5
Total no. of clones

6

7

8

+ (9)

+ (4)

–

+ (6)

+ (1)

+ (3)

+ (3)

13

+ (14)

–

–

–

14

+ (12)

+ (1)

+ (2)

–

15

32

12

9

3

14
13

The CP region (705 nt) of ASPV genome was amplified by RT-PCR and the DNA products were cloned and sequenced.

(Figure 4). We then extracted RNA from the peel and flesh
tissues of fruit showing cracks on the apple seedling No. 1, andsubjected the RNA to RT-PCR, in which we found that ASPV
was distributed in the fruit showing cracks. Cloning and sequence
analysis of the amplified DNA products from the fruit indicatedthat twenty DNA clones from the fruit tissues had the same
sequence as GCSPV2, whereas GCSPV6 was not detected in
the fruit tissues. Furthermore, apple seedlings Nos. 2, 3, and5 infected with GCSPV6 showed no symptoms on the fruits(Table 4 and Figure 4). These results strongly suggest that oneof the ASPV sequence variants, namely GCSPV2, probably serves
as one of the causal agents of AGCD.
We have determined the complete nucleotide sequences of
the GCSPV2 genomes using DNA clones amplified from a fruit
showing symptoms. The genome of GCSPV2 consists of 9294 nt(accession LC533839) excluding the 30 -poly A tail and encodes
five proteins from the 50 -terminus; an RNA replication-associatedprotein (Rep) (248 kDa, 2018 aa), triple gene block (TGB)
proteins [TGB1 (25 kDa, 223 aa), TGB2 (13 kDa, 120 aa), TGB3
(8 kDa, 75 aa)] and a CP (42 kDa, 396 aa). Phylogenetic analysis
based on the complete nucleotide sequence of 28 isolates of ASPV,
2 of AGCaV, and 5 species of other foveaviruses showed that these
ASPV isolates (including AGCaV) could be grouped into 4 clades(I ⇠ IV), and that GCSPV2 was most closely related to IF38 froman apple in clade III (Figure 5). The identity of the complete
nucleotide sequence between GCSPV2 and IF38 was 93.7% and
the identities of the amino acid sequences between GCSPV2 andIF38 were 97.8% (Rep), 97.8% (TGB1), 97.5% (TGB2), 100%
(TGB3), and 96.7% (CP). ASPV-IF38 was originally isolated from‘Fuji’ apple in Iwate University. Morioka, Japan which is not

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affected with AGCD (Yoshikawa et al., 2001). The nucleotide
sequence identities of the genomes between ASPV-GCSPV2 and
AGCaV-aurora were 76.6%, and their amino acid identities
between them were 88.8% (Rep), 90.6 (TGB1), 84.2% (TGB2),
84.5% (TGB3), and 76.2% (CP).

DISCUSSION
To investigate viruses in fruit trees, interest in using
comprehensive NGS analysis has been increasing in recent
years (Coetzee et al., 2010; Ho and Tzanetakis, 2014; Rott et al.,2017; Maliogka et al., 2018). Virome analysis using NGS is
expected to be a technique used to search for unknown viralpathogens (Ho and Tzanetakis, 2014; Pecman et al., 2017). For
this reason, we first conducted a virome analysis of diseased
apple trees in order to determine the types of viruses present
in apple trees affected by ARRD and AGCD. Polyacrylamide
gel electrophoresis of the dsRNAs extracted from ARRD- andAGCD-infected samples showed several high molecular weightbands as shown in Supplementary Figure S2, which is consistent
with the dsRNA patterns of plants infected with ACLSV, ASGV,or ASPV, as reported previously (Yoshikawa and Takahashi, 1988;
Magome et al., 1997a; Yoshikawa, 2008). In virome analysis ofdsRNAs from diseased samples, we found that five known viruses(ACLSV, ASGV, ASPV, AGCaV, and ApLV) have been infecting
both ARRD and AGCD apple trees, and that no new viruses
were detected in either type of diseased apples. It was reported
that all apple trees with AGCD are infected with ACLSV, ASGV,
and/or ASPV (Pacific Northwest Plant Disease Management

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FIGURE 4 | Apple seedlings inoculated with RNA transcripts of ASPV and ALSV-AtFT/MdTFL1 about 10 months after inoculation (upper left) and fruits produced on
the inoculated apple seedlings. Control; infected with ALSV-AtFT/MdTFL1 only, No. 1; with GCSPV2 and GCSPV6, No. 2; with GCSPV6 and GCSPV7, No. 3; withGCSPV5, GCSPV6, GCSPV7, and GCSPV8, No. 4 with GCSPV5.

Handbook2 ; Viral diseases, WUS Tree Fruit3 ). Deep sequencing
analysis of an apple sample from Korea showed that these fiveviruses were identified from eight apple samples showing growth
retardation (Cho et al., 2016). In Japan, ACLSV, ASGV, and
ASPV have been reported as causative viruses of the top working
diseases in apple fruits growing on crab apples (Malus prunifolia
var. ringo, M. sieboldii, and M. sieboldii arborescens) as rootstocks(Yanase, 1974). On the other hand, most domesticated orchard
apple plants (M. domestica) were latently infected with ACLSV,ASGV, and/or ASPV and these are widely distributed as latentviruses in cultivated apples in Japan.
To investigate the association between a specific disease anda particular virus, it is necessary to isolate each virus fromthe diseased apple trees, and to infect each virus into separate
herbaceous plants. In general, leaf extracts of diseased samples
were sap-inoculated to different test plants for the separationof target viruses. However, it is difficult to inoculate each apple
virus into separate plants because the available hosts (C quinoa
and N. occidentalis etc.) of apple viruses are common for twoor three apple viruses (Yanase, 1974; Koganezawa and Yanase,1990). For that reason, we amplified the full-length DNAs ofviral genomic RNAs by RT-PCR using primers containing the
2
3

T3 promoter sequence, and the full-length viral RNAs weretranscribed in vitro from the amplified DNAs as templates(Supplementary Figure S3). Inoculation of RNA transcripts
into herbaceous plants resulted in a high infection rate (50–
100% depending on virus), and we were able to infect eachvirus coexisting in an ARRD-infected apple tree into separate
herbaceous plants (Supplementary Figure S3). This method ofusing in vitro viral RNA transcripts is a very effective method for
isolating and separating each virus from fruit trees with multiple
viruses, if the entire genome sequences of the target viruses areavailable. Because the apple russet ring disease is implicated tobe caused by a strain (or by strains) of ACLSV (van der Meer,
1986; Desvignes and Boye, 1988; Wood, 2001; Howell et al.,2011), in our subsequent experiments, we considered ACLSV asa candidate for the ARRD causal virus.
For the identification of the pathogenic viruses of diseases,
it is important to demonstrate not only the viral species but
also their SVs in the diseased trees. The present study indicatedthat at least seven SVs of ACLSV were found in leaf samples of
ARRD-apple (PK-51) (Table 2, Figure 1, and Supplementary
Figure S5). Though the data are not shown, multiple SVs werealso found for ASGV and ASPV in the ARRD-apple. Similarly,
at least eight SVs of ASPV were detected in the leaf samples of
AGCD-apple (P-190) (Table 3 and Supplementary Figure S8).
Just as multiple virus species gathered in a single tree, eachhttps://pnwhandbooks.org/node/2133
http://treefruit.wsu.edu/web-article/viral-diseases/

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FIGURE 5 | Phylogenetic tree based on the complete nucleotide sequences of ASPV isolates, AGCaV isolates, and five other foveaviruses including grapevine
rupestris stem pitting associated virus (GRSPaV), apricot latent virus (ApLV), grapevine virus T (GVT), peach chlorotic mottle virus (PCMV), and cherry virus B (ChVB).
A sequence variant analyzed in this study is shown in red. The tree was generated by CLUSTALW program (DNA databank of JAPAN [DDBJ]; URL:
http://clustalw.ddbj.nig.ac.jp/). Bootstrap values for each node are shown (1000 replicates).

SV in the same virus is likely to have been brought into asingle tree by grafting widely used in cultivation of fruit trees
in Japan (Magome et al., 1997b). We previously reported theuse of identical ALSV labeled with yellow and cyan fluorescence
proteins (YFP and GFP) replicated predominantly in discrete
areas and separately distributed in leaves coinfected with ALSVYFP and ALSV-CFP (Takahashi et al., 2007). Spatial separation
was also found in plants co-infected with bean yellow mosaic

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virus (BYMV)-YFP and BYMV-CFP (Takahashi et al., 2007) andplum pox virus (Dietrich and Maiss, 2003), indicating that the
separate distribution of virus population may be a universal
phenomenon among the same virus species. Considering the
distribution of SVs of apple viruses in apple plants, it is assumed
that many SVs of each virus are spatially separated from eachother in either mosaic or patch form within the tissues andleaves of infected trees. In PK-51 affected by ARRD, there are

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obvious year-to-year fluctuations in which symptoms appear only
for a few fruits in a shoot, or with a relatively large number ofsymptoms appearing in the same tree. Although temperaturesduring spring to early summer are reported to have a significant
effect on the expression of fruit symptoms (Wood, 1971), anotherreason for this fluctuation may be due to the interaction between
sequence variants coexisted in the infected tree. That is, if
the pathogenic variant predominates, symptoms will appear;
otherwise, it will not appear at all.
In this study, we reported that one of the SVs of ACLSV(RRACV2), but not other SVs (RRACV1, RRACV3, and
RRACV5), caused leaf and fruit symptoms characteristic of
ARRD in apple trees. van der Meer (1986) reported that
ACLSV isolates from ARRD-apples were graft-transmitted from
C. amaranticolor to the apple seedlings and some isolates induced
russet rings on apple cultivars. He also mentioned that clearly
different ACLSV strains can be obtained from the same tree.
Desvignes and Boye (1988) also reported that russet rings are very
likely to be caused by particular isolates of ACLSV, because backinoculation from C. quinoa to ‘Golden Delicious’ was positive.
Although these two reports did not characterize ACLSV as asingle isolate, the results obtained in this study are consistent
with those reports. Wood (2001) reported that at least six distinct
strains or forms appear to be present, assuming that all of therusset ring disorders found in New Zealand apple trees are
related. Further study is needed to determine whether ACLSVRRACV2 alone causes ARRD, or if other isolates/strains also
cause ARRD. An isolate B6 of ACLSV was isolated from an
ARRD-apple different from PK-51 (Yaegashi et al., 2007), and B6
is very closely related to ACLSV-RRACV2 in the phylogenetic
tree (Figure 3). It would be interesting to test if B6 induces
ARRD in apple trees. As most ACLSV strains or isolates do notcause ARRD in apple (Yanase, 1974; Yaegashi et al., 2011), it isimportant to identify which genes, or nucleotides, and amino acidsequences in the ACLSV-RRACV2 genome are involved in the
induction of leaf and fruit symptoms characteristic to ARRD.
For AGCD, it is assumed that ASPV or AGCaV may be acausal virus (Howell et al., 2011; James et al., 2013); however,there are no reports of back-inoculation of the isolated virus
to apple plants. In AGCD, the symptoms appear only on the
fruits and not on the leaves, so we must observe the fruitsof the back-inoculated apple plants. In this study, we used
VIF technology using the ALSV vector to accelerate apple fruit
formation, because it takes at least 6–7 years for apple seedlings
to blossom. ALSV does not induce any symptoms on leaves andfruits (Yamagishi et al., 2014).
The apple tree (P-190) affected by AGCD tested in thisstudy showed symptoms in almost all fruits every year. Our
results indicated that the apple tree (P-190) with AGCD has
a complex viral infection because the plant contained at least
eight SVs (GCSPV1- GCSPV8) (Table 3), even for ASPV alone.
The type and proportion of SVs were different among the
inoculated plants, even when using the same RNA transcript as
inoculum (Table 4). We showed that one of the sequence variants(GCSPV2) probably induces severe cracks on the fruit surface(apple seedling No. 1 in Figure 4), which is very similar to the
AGCD symptoms shown in Supplementary Figure S1. Though

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the apple seedling No. 1 was infected with both GCSPV2 andGCSPV6 (Table 4), most of the variants detected were GCSPV2(13/14 clones) (Table 4) and only GCSPV2 was detected fromfruit showing cracks (20/20 clones). Furthermore, apple plants
infected with other SVs including GCSPV6 produced normalshaped apple fruits (Table 4 and Figure 4). ASPV-GCSPV2 wasdistributed in the fruit and showed cracks, strongly suggestingthat ASPV-GCSPV2 might be one of the AGCD causal agents.
We plan to inoculate the virus-free ‘Golden Delicious’ trees with
branches of infected trees (No. 1 to No. 5) by grafting in ARRD
to further confirm the ASPV-SVs pathogenicity this year.
Since Jelkmann (1994) first reported the nucleotide sequenceof the pear ASPV genome, the complete nucleotide sequences ofmany isolates from apple, pear, and quince have been reported, as
shown in Figure 5. There is a great genetic diversity among ASPV
isolates, and some isolates had less than about 72% nt identity (or80% aa identity) between their CP or polymerase genes, whichcorresponds to the species demarcation criteria in the Foveavirus
genus (Youssef et al., 2011; Adams et al., 2012). James et al.
(2013) reported that a putative new foveavirus (AGCaV), or avariant or strain of ASPV was isolated from an Aurora Golden
Gala apple showing severe symptoms of AGCD. A phylogenetic
analysis based on the complete nucleotide sequences showed that
ASPV isolates might be grouped into 4 clades, and that GCSPV2
was in clade III with IF38 from apple (Figure 3). On the other
hand, AGCaV (aurora-1) was grouped into clade II (Figure 3),
suggesting that multiple ASPV variants including AGCaV may
cause AGCD. It will be interesting to determine which gene(s)in the ASPV genome are involved in the induction of fruit
symptoms of AGCD.
As previously mentioned, there are virus-like diseases ofdeciduous fruit trees whose causal agents are still not identified.
The strategy for fulfillment of the Koch’s Postulates presented
here (Supplementary Figure S9) can provide a system to prove
if the virus found in diseased tissues is the same as the pathogen
causing diseases in fruit trees.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories
and accession number(s) can be found in the article/
Supplementary Material.

AUTHOR CONTRIBUTIONS
CL amplified the full-length DNA, transcribed infectious RNAs,
and analyzed the sequence variants of ASPV. HY conducted aphylogenetic analysis of ACLSV and ASPV. RK amplified thefull-length DNA, transcribed infectious RNAs, and analyzed thesequence variants of ACLSV. AK analyzed the viruses of apple
plants back-inoculated with ACLSV and ASPV and determinedthe complete nucleotide sequence of the ASPV-GCSPV2 genome.
NYa carried out the inoculation of virus RNA to apple plants byparticle bombardment and determined the complete nucleotide
sequences of the ACLSV-RRACV1 and RRACV2 genome. TI

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Apple RR and GC Diseases

ACKNOWLEDGMENTS

conducted the graft-transmission tests. NYo is the principal
investigator, and he supervised the experiments and wrote themanuscript. All the authors contributed to the article andapproved the submitted version.

We would like to thank Mr. Yudai Kikuchi for technical assistant.

SUPPLEMENTARY MATERIALFUNDING

The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fmicb.
2020.01627/full#supplementary-material

This work was supported by JSPS KAKENHI Grant
Number 24380027.

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Conflict of Interest: The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interest.
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