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Journal of Pathology (2013), 95 (2), 221-235 Edizioni ETS Pisa, 2013 221

INVITED REVIEW OF KIWIFRUIT (ACTINIDIA SPECIES)

A.G. Blouin1, M.N. Pearson2, R.R. Chavan2, E.N.Y. Woo2, B.S.M. Lebas3, S. Veerakone3, C. Ratti4, R. Biccheri4, R.M. MacDiarmid1,2 and D. Cohen1

1The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, New Zealand 2School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand 3Plant Health and Environment Laboratory, Ministry for Primary Industries, PO Box 2095, Auckland 1140, New Zealand 4Dipartimento di Scienze Agrarie, Area Patologia Vegetale, Viale G. Fanin 40, 40127 Bologna, Italy

SUMMARY bark cracking and cane wilting. Pelargonium zonate spot (PZSV) has been detected in Italy associated with Kiwifruit (Actinidia deliciosa) was introduced to New severe symptoms on leaves and fruit. Zealand more than one hundred years ago and the New Zealand-raised cv. Hayward is now the dominant culti- var grown worldwide. Further accessions of kiwifruit INTRODUCTION seed and scionwood have been sourced from China for research and breeding. In one importation consign- In 1904, Isabel Fraser introduced the first kiwifruit ment, the first virus naturally infecting kiwifruit, Apple seed to New Zealand, and by 1910 the raised by a stem grooving virus (ASGV), was identified following friend, Alexander Allison, produced the first fruit out- symptoms observed in quarantined plants (2003). Since side China (Ferguson and Bollard, 1990). Actinidia deli- that time a further 12 viruses have been identified in ki- ciosa cv. Hayward was selected around 1925 and ki- wifruit. We classify these 13 viruses into three groups. wifruit production started in New Zealand by 1930 The first group comprises the non-specialist viruses (Ferguson and Bollard, 1990). The original name ‘Chi- and includes Alfalfa mosaic virus (AMV) and Cucumber nese gooseberry’ was replaced by ‘kiwifruit’ when the mosaic virus (CMV) both members of the family Bro- first fruit were exported to the USA in 1959 (Ferguson moviridae. The group also includes a further five viruses and Bollard, 1990). The name ‘kiwifruit’ is now often that appear to have limited effect on kiwifruit: two to- used for all species within the genus Actinidia. Until bamoviruses, Ribgrass mosaic virus (RMV) and Turnip 2000 A. deliciosa ‘Hayward’ was the cultivar of choice, vein clearing virus (TVCV); a tombusvirus, Cucumber and almost all the international trade in kiwifruit was of necrosis virus (CNV); a novel potexvirus; and Apple this one cultivar. When facing overproduction in the stem gooving virus (ASGV, genus Capillovirus). Most of early 1990s, the New Zealand industry innovated and the viruses classified in this first group are cosmopolitan assessed the commercial potential of another species, and sometimes orchard weeds provide reservoirs for in- Actinidia chinensis (Ferguson and Huang, 2007). fection. A. deliciosa has fruit with green flesh and hairy skin, The second group comprises the kiwifruit-adapted while A. chinensis has smooth-skinned fruit and, usually, viruses. This group includes three novel viruses. i.e. two yellow flesh. Other differences include fruit flavour, vitiviruses, Actinidia virus A (AcVA) and Actinidia virus flower size, shoot hairiness, geographic distribution, B (AcVB), and a citrivirus closely related to Citrus leaf chromosome number, and leaf shape (Ferguson and blotch virus (CLBV). In addition, preliminary evidence Bollard, 1990). The introduction of the yellow-fleshed of a novel virus belonging to the family A. chinensis cv. Hort16A, marketed under the Zespri has been obtained. Gold Kiwifruit brand, changed the industry by offering The third group of viruses induces disease in ki- a product that complemented, rather than competed wifruit. To date only two viruses have caused significant with, cv. Hayward resulting in increased consumption damage to kiwifruit within commercial orchards. In (Anonymous, 2012). Since 2000, most newly planted or- New Zealand, Cherry leaf roll virus (CLRV) has been chards in New Zealand have been A. chinensis and cv. detected on kiwifruit associated with symptoms includ- Hort16A now represents about 26% of the New ing leaf spots, fruit malformation, reduction in yield, Zealand export of kiwifruit (Anonymous, 2012). Other yellow-fleshed A. chinensis have now been commer- cialised in a number of countries, including cvs Jintao or ENZAGold™. About 30% of kiwifruit planted in China is now A. chinensis (Ferguson and Seal, 2008). Corresponding author: A.G. Blouin The success of the yellow and, subsequently, a red- Fax: +64.9.9257001 E-mail: [email protected] fleshed A. chinensis, combined with the need to intro- 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 222

222 Viruses of kiwifruit Journal of Plant Pathology (2013), 95 (2), 221-235

Fig. 1. Kiwifruit production worldwide past 20 years (after Belrose Inc., 2012). Data from world kiwifruit review 2012 based on predicted production for 2012. Production for 1999-2002 is in dark grey with a total production of 1,152,578 tons. The predicted production for 2009-2012 is in light grey with a total of 1,862,487 tons.

duce novelty into the market, has intensified breeding all cv. Hort16A plantings in New Zealand, and possibly programmes in New Zealand, Italy and China. This in other producing countries, could be removed be- breeding activity has resulted in more plant movement cause of the cultivar’s vulnerability to the disease between countries. Despite the development of new va- (Anonymous, 2012; Young, 2012) resulting in an urgent rieties, ‘Hayward’ is still the predominant fruit traded need for new resistant cultivars. internationally, comprising an estimated 90–95% of the Although there were some reports of virus-like symp- worldwide kiwifruit market (Ferguson and Seal, 2008). toms, no viruses were identified in kiwifruit before International kiwifruit production is concentrated in 2003. The first indication of a kiwifruit-infecting virus relatively few countries. The top four countries are Chi- comes from New Zealand quarantine records in 1983. na, Italy, New Zealand and Chile, which collectively Gary Wood, from the then Department of Industrial produce more than 80% of the world’s kiwifruit crop; and Scientific Research (DSIR, New Zealand), docu- the top ten producing countries represent more than mented local lesions observed on Chenopodium quinoa 96% of the world supply (Anonymous, 2012) (Fig. 1). after sap inoculation of kiwifruit imported from China Commercial production of kiwifruit in China has in- and held in quarantine. The infected kiwifruit plants creased steadily over the past two decades; now, China were either destroyed or died during thermotherapy (G. is the biggest producer, with more than 25% of the Wood, personal communication). world’s production. This has contributed to the 62% in- In the 1980s, as Italy was becoming an important ki- crease in total world production over the past 10 years wifruit producer with the second greatest area planted (Anonymous, 2012). worldwide, there were no records of viruses infecting Disease pressure is a new concept for the kiwifruit the crop. Caciagli and Lovisolo (1987) surveyed com- industry. Some fungal diseases were reported previously, mercial orchards for potential viral diseases and collect- such as Armillaria novae-zelandii in New Zealand ed samples from 100 symptomless A. deliciosa and one (Horner, 1992); Phomopsis sp. in Greece (Elena, 2009); plant of A. deliciosa that showed chlorotic mottling. The Cadophora melinii in Italy (Prodi et al., 2008); and verti- extracts from these plants were mechanically inoculated cillium wilt of gold kiwifruit in Chile (Auger et al., into four herbaceous indicators (C. quinoa, C. amaranti- 2009). To date, these pathogens tend to be localised. color, glutinosa and N. clevelandii). None of The recent detection of a virulent strain of the 404 inoculated indicator plants displayed symptoms. Pseudomonas syringae pv. actinidiae in Italy and New Additionally, the authors challenged young A. deliciosa Zealand (Ferrante and Scortichini, 2010; Everett et al., plants with 17 common viruses from Italy, including Al- 2011) has had disastrous consequences for the produc- falfa mosaic virus (AMV) and Cucumber mosaic virus tion of A. chinensis cv. Hort16A. Some anticipate that (CMV). Only three viruses, Tobacco necrosis virus 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 223

Journal of Plant Pathology (2013), 95 (2), 221-235 Blouin et al. 223

(TNV), Tobacco rattle virus (TRV) and CMV, induced identified in kiwifruit was detected by leaf symptoms, symptoms on the inoculated leaves of the kiwifruit, and transmission electron microscopy (TEM) and mechani- only CMV moved systemically. The authors concluded cal transmission to herbaceous indicators, and identified that kiwifruit may be resistant to virus infections. by DAS-ELISA, RT-PCR and sequencing of amplicons. A few years later, during a survey in the Fujian Other kiwifruit from the same consignment were subse- Province in China, Lin and Gao (1995) identified one quently studied further and new viruses were identified. plant showing a “mosaic disease” attributed to an To date, the viruses discovered in kiwifruit can be di- unidentified virus. Nitta and Ogasawara (1997) report- vided in three groups. The first group comprises AMV, ed evidence of a graft-transmissible agent causing virus- ASGV, CMV, Cucumber necrosis virus (CNV), Ribgrass like symptoms. Using cuttings from Actinidia polygama mosaic virus (RMV), Turnip vein clearing virus (TVCV), plants collected in the mountains of Hiroshima Prefec- and a novel potexvirus, tentatively named Actinidia virus ture (Japan) as rootstocks, they observed chlorotic spots X (AVX). These viruses are mostly ubiquitous/ cosmopol- and rings on the eight different A. deliciosa varieties itan and, so far, do not show a detrimental effect on com- used as male scions. In neither case was the causal agent mercial kiwifruit. Most of these viruses are distributed identified. worldwide over a large host range and have been detected In 2003, Apple stem grooving virus (ASGV) was iden- in alternative hosts neighbouring kiwifruit orchards. tified in a kiwifruit import from China held in New The second group comprises the putatively kiwifruit- Zealand quarantine (Clover et al., 2003). This first virus specific viruses that, to date, are only known to have

Fig. 2. A. Symptomatic leaf of Actinidia glaucophyla infected with Alfalfa mosaic virus. B. Symptomatic leaf of Actinidia chinensis infected with Actinidia virus A, Actinidia virus B and Actinidia citrivirus. C. Symptomatic Nicotiana glutinosa infected with Ac- tinidia citrivirus. D. Symptoms associated with Cherry leaf roll virus in Actinidia chinensis cv. Hort16A. Chlorosis developing into necrosis on a leaf. E. Symptoms associated with Cherry leaf roll virus (CLRV) in Actinidia chinensis cv. Hort16A, a regular fruit on the left with a beak at the calyx end characteristic of cv. Hort16A, and fruit infected with CLRV on the right not showing the beak. F. Symptoms observed on leaves of Actinidia chinensis cv. Hort16A infected with Pelargonium zonate spot virus.. 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 224

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this single host or are likely to have a very limited host The third and most concerning group includes two range. In this group we have identified two vitiviruses, viruses that have very recently been detected in ki- Actinidia virus A (AcVA) and Actinidia virus B (AcVB), wifruit. Cherry leaf roll virus (CLRV) in New Zealand and a citrivirus closely related to Citrus leaf blotch virus and Pelargonium zonate spot virus (PZSV) in Italy both (CLBV). There is also evidence of a novel virus from the cause severe damage to the commercial crop. Almost 10 family Closteroviridae, although the data for this virus years since the first publication of kiwifruit virology, we are still being collected. describe now the 13 viruses detected in kiwifruit to

Table 1. Viruses infecting Actinidia species in nature: taxonomic allocation, epidemiology, geographical distribution and reference to kiwifruit.

Geographical Virus name, Symptoms on Particle size Host range distribution in First report in (abbreviation), Kiwifruit, vectors kiwifruit kiwifruit Genus, Family (other hosts) Actinidia citrivirus, Flexuous, 750–800 Associated with vein Kiwifruit China, New Pearson et al. Citrivirus, nm clearing and mild Zealand (2011) mottling on leaves and interveinal chlorosis. No known vector. Actinidia virus A Flexuous, 750–800 Associated with leaf Kiwifruit China, Italy, New Blouin et al. (2012) (AcVA) and nm clearing, ringspots but Zealand Actinidia virus B can be latent. No (AcVB), Vitivirus, known vector. Betaflexiviridae Actinidia virus X Flexuous, 470-580 Can be latent. Vector Unknown New Zealand Pearson et al. (AVX), Potexvirus, nm unknown. (2011) Alfaflexiviridae Apple stem 620-700x12 nm Interveinal mottling, Apple, pear, China, New Clover et al. (2003) grooving virus, Actinidia isolate = chlorotic mosaics and cherry, citrus and Zealand (ASGV), 680 nm ring-spots. No known kiwifruit and nine (worldwide) Capillovirus, vector. dicotyledonous Betaflexiviridae families Alfalfa mosaic virus Baciliform of Mild symptoms on A. Very wide host New Zealand Pearson et al. (AMV) different length chinensis. Transmitted range (worldwide) (2011) Alfamovirus (56, 43, 35 and 30 by aphids, seeds and nm) and constant pollen. diameter of 18 nm Cherry leaf roll Isometric particles Necrotic spot on leaf, Wide host range New Zealand Woo et al. (2012b) virus ca. 28 nm in bark craking and cane (worldwide) (CLRV) diameter wilting in severe Nepovirus infection. Cv. Zespri Gold Kiwifruit shape altered. Transmission through seed, pollen, grafting and mechanical inoculation. No known vector. Cucumber mosaic Isometric ca. 28 Chlorosis on A. Extremely wide Italy, New Zealand Pearson et al. virus, (CMV) nm in diameter chinensis. Transmitted (worldwide) (2009) Cucumovirus, by aphids, seeds and Bromoviridae pollen. Cucumber necrosis Isometric, 31 nm Symptomless in Cucumber, lettuce, China, Italy, New Lebas et al. virus, (CNV) in diameter kiwifruit. Vectored by tomato and Zealand (unpublished) Tombusvirus, fungus Olpidium kiwifruit (Canada, the USA, radicale. China, Italy, New Not seed transmitted. Zealand) Pelargonium zonate Quasi-spherical, Concentric Large host range Italy Biccheri et al. spot virus (PZSV), non-enveloped and chlorotic/necrotic rings including (Australia, France, (2012) Anulavirus, diameter ranging and line patterns. pelargonium, Italy, Israel, Spain, Bromoviridae from 25 to 35 nm Transmitted by seed tomato, pepper, and the USA) and pollen. artichoke, and kiwifruit Ribgrass mosaic 300 x 18 nm rigid Can be symptomless in Wide host range China, New Chavan et al. virus, (RMV), rod kiwifruit. Zealand (2009) Turnip vein No known vectors, (worldwide) clearing virus mechanically (TVCV), transmissible, possibly Tobamovirus, transmitted on seed. 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 225

Journal of Plant Pathology (2013), 95 (2), 221-235 Blouin et al. 225

Table 2. Diagnostic tools: reagents for ELISA when available, and primers used and conditions for PCR assays.

PCR Virus name ELISA Annealing Amplicon Forward primer Reverse primer Reference (°C) size (bp) Actinidia Dweet mottle antiserum CLBV 1F CLBV 5R 58 425 Chavan et al. citrivirus Antiserum USDA253, AGCCATAGTTGAACCATTCCTC GCAGATCATTCACCACATGC (unpublished) (courtesy Dr. Richard Lee) AcVA Not available AcVA 1F AcV 1R 55 269 Blouin et al. ATGATGGGGTGTTCTATGGGTGG CTCATTCTCCAMCCRCARAAGAG (2012) CT AcVB Not available AcVB 1F AcV 1R 55 529 Blouin et al. AATTCGGACCACTCCTGAGGC CTCATTCTCCAMCCRCARAAGAG (2012) AMV Bioreba (Switzerland) Cat AMV for AMV rev 55 415 Blouin et al. 140512-140522 TGTCTCACTGATGACGTG CATACCTTGACCTTAATCCAC (2010) Only reliable for symptomatic Actinidia tissue and herbaceous indicators ASGV Bioreba Cat 150912 and CTLV-AP CTLV-AM 60 456 Ito et al. 150922 CCTGAATTGAAAACCTTTGCTGCC TAGAAAAACCACACTAACCCGG (2002) ACTT AAATGC

AVX Rabbit polyclonal antiserum AVX-F (3963) AVX-R (4118) 58 175 D. Cohen and saised against purified virus AAGTCCGCAACACCTACCTG GGACAGACGATAGCAGCCTT A.G. Blouin (Plant and Food Research) (unpublished) CLRV Bioreba, Cat 150822 and CLRV-F CLRV-R 55 416 Werner et al. 150812 TGGCGACCGTGTAACGGCA GTCGGAAAGATTACGTAAAAGG (1997)

CMV Bioreba, Cat 160612 and CMV-F CMV-R 54 885 Felix and Clara 160622 CTTTCTCATGGATGCTTCTC GCCGTAAGCTGGATGGAC (2008)

CMV nF (nested if required) CMV nR (nested if required) Nested: ACTATTAACCACCCAACCT TTTGAATGCGCGAAACAAG 172

CNV DSMZ (Germany), antisera PCR1 PCR1 PCR1 PCR1 PCR1 AS-0130 Gral. Tombusvirus F1 Gral. Tombusvirus R1 55 587 Harris et al. AAGGGTAAGGATGGTGAGGA TTTGGTAGGTTGTGGAGTGC (2007)

CuNV-F791 (nested) CuNV-R1002 (nested) Nested- Nested- Nested-PCR CCTCGCAGAAGACCTTATGC GCCGACTCCTCCACTCCA PCR PCR Lebas et al. 60 215 (unpublished) PZSV ADGEN Phytodiagnostics PZSV2 F PZSV2 R 55 997 Ratti et al. GATAAATTCAGAGCTCTCGG ATCTCTGCAGATTGTGTTCC (unpublished) RMV and Rabbit polyclonal antiserum AT2F AT 4R Chavan et al. TVCV raised against purified TMV AGACAGCAATTCTCAAACTTGT CGGTCGCATCATCAACAC 55 223 (unpublished) (Auckland University)

date. This represents the first review of kiwifruit viruses, is the type member of the genus Alfamovirus and has including images of symptoms (Fig. 2), a summary table four bacilliform type particles (Fauquet et al., 2005). of each virus (Table 1), and a summary of diagnostic CMV is the type member of the genus Cucumovirus and tools including primer sequences and amplification con- has icosahedral particles. ditions (Table 2). AMV was one of the first viruses detected and identi- fied in kiwifruit in New Zealand (Pearson et al., 2009). It was first detected in Actinidia glaucophylla, showing NON-SPECIALIST VIRUSES strong yellow mosaic patterns (Fig. 2A). Extracts from the chlorotic blotch easily transmitted the virus to a Alfalfa mosaic virus and Cucumber mosaic virus. range of herbaceous indicator plants. In the same AMV and CMV are two viruses infecting a very broad germplasm collection, AMV was also isolated from Ac- host range, with over 1200 plant host species in over tinidia guilinensis and A. fortunatii showing mottled and 100 families for CMV (Douine et al., 1979) and 300 generally chlorotic leaves. In these hosts, the plants species in 22 plant families for AMV (Hull, 1969). The looked unthrifty and the virus symptoms were wide- addition of Actinidia species to their host range is not spread in the block. The symptoms were observed in unexpected. Because of the damage CMV causes on spring for four consecutive years. AMV and CMV have some economically important crops, it was included in been found as a dual infection in both A. glaucophylla the “Top 10 plant viruses” in a recent molecular pathol- and A. fortunatii, and CMV was also detected in a single ogy review (Scholthof et al., 2011). Both viruses belong symptomless infection of A. glaucophylla. to the family Bromoviridae and are efficiently vectored AMV has only been detected once in A. chinensis in by a number of aphid species. They are also transmitted New Zealand. The plant showed a few leaves with very by seed and are easily transmissible mechanically. AMV minor chlorosis and the symptoms could not be ob- 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 226

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served the following year. Inoculation of AMV to A. chi- quencing of the amplicons. nensis seedlings induced foliar symptoms on one or two Symptoms on A. chinensis include chlorosis of leaf leaves above the inoculated leaf, but newer leaves were veins and adjacent tissue during spring and chlorotic symptomless. CMV has been detected in Italy on one A. mottles, mosaics, and ringspots during summer. Symp- chinensis plant with pale mottling of the leaves. toms on A. deliciosa include chlorotic mottling or mo- AMV and CMV can be detected by RT-PCR in Ac- saic during spring and ringspots during summer months tinidia sp. (Table 2). DAS-ELISA can also be used for (Chavan et al., 2009). Some of the symptoms resemble both viruses but AMV can only be detected in sympto- those previously described in Actinidia infected with matic tissues. Both viruses are readily transmissible to a ASGV (Clover et al., 2003) and subsequent investiga- range of herbaceous indicators including N. benthami- tion has established that most of the plants were co-in- ana, N. clevelandii, N. glutinosa, and N. occidentalis. fected with other viruses (Chavan et al., unpublished These two viruses are similar in terms of their abun- information). Symptoms on mechanically inoculated in- dance in the surrounding weeds, and also by sharing the dicators include local chlorotic lesions in C. amaranticol- same vectors. Both are present worldwide and are likely or and C. quinoa, systemic mosaic and distortion in N. to infect Actinidia sp. causing some concerns for the benthamiana, systemic necrotic ringspots and chlorotic non-commercial species (A. glaucophylla, A. guilinensis vein banding and dark green blistering and distortion in and A. fortunatii). Fortunately, the viruses do not ap- N. clevelandii, local necrotic lesions and systemic mottle pear to have a detrimental effect on either A. chinensis in N. glutinosa and N. occidentalis, and mild systemic or A. deliciosa. Their impact on these important crops is mottle in Phaseolus vulgaris (Chavan et al., 2009), but therefore negligible. some of these symptoms may be caused by co-infecting viruses. Ribgrass mosaic virus and Turnip vein clearing For routine diagnosis, RMV and/or TVCV can be virus. RMV and TVCV are two closely related species detected in Actinidia leaf samples by conventional RT- in subgroup 3 of the genus Tobamovirus, family Vir- PCR (Table 2). ELISA, using a rabbit polyclonal anti- gaviridae. Both viruses have 300 nm rod-shaped parti- serum raised against purified TMV (M. Pearson, The cles with positive sense, single-stranded RNA (ssRNA) University of Auckland), detected Actinidia isolates of (Adams et al., 2009). RMV was first reported from Plan- RMV in herbaceous indicators but failed to detect the tago (Holmes, 1941) and has been variously referred as virus in infected A. chinensis and A. deliciosa plants Holmes ribgrass virus, -ribgrass (Chavan et al., 2009). There are no known arthropod strain, Crucifer TMV, and TMV Wasabi (Gibbs, 1999). vectors of tobamoviruses but they can survive in sap for It has been reported from at least 67 different species prolonged periods (Oshima and Harrison, 1975). To- belonging to 15 diverse dicotyledonous and mono- bamoviruses are highly infectious and readily spread by cotyledonous families (Chavan et al., 2012). Symptoms contact between infected and healthy plants or via ma- include systemic chlorotic mottling, ring-like markings, chinery and human handling (Gibbs, 1977). Conse- chlorotic streaks along the veins and twisting of the quently, similar treatments to those recommended to petioles in Plantago species, vein clearing in turnip prevent the spread of TMV, such as seed sterilisation us- (Lartey et al., 1993), necrotic mosaic in tobacco and in- ing hypochlorite, should be used to prevent virus on ternal browning of tomato fruit (Oshima and Harrison, seed coats from infecting seedlings during nursery oper- 1975). Tobamoviruses have no known natural vectors ations (Cohen et al., unpublished information). Overall, but the particles are stable and readily mechanically RMV and TVCV do not appear to cause significant transmitted. They can also be carried and transmitted damage to commercial kiwifruit orchards. from the surface of seeds (Gibbs, 1977). RMV was first detected in A. deliciosa and A. chinen- Apple stem grooving virus. ASGV is the type mem- sis held in post-entry quarantine in New Zealand (Cha- ber of the genus Capillovirus, family Betaflexiviridae. Its van et al., 2009) and the complete sequences of the iso- genome consists of a positive-sense ssRNA of 6,496 nu- lates from A. chinensis (GenBank accession No. cleotides (excluding the polyA-tail) enveloped in a flex- GQ401366.1) and A. deliciosa (GQ401365.1) were sub- uous, filamentous particle of 620-700x12 nm sequently published (Chavan et al., 2012). RMV and (Yoshikawa, 2000). Citrus tatter leaf virus (CTLV) is re- TVCV were first reported in New Zealand from Planta- garded as an isolate of ASGV, being indistinguishable go spp. (Cohen et al., 2012). Subsequent studies have from it biologically, serologically and in genome organi- identified both viruses in A. chinensis in New Zealand, zation (Yoshikawa, 2000). The main crop hosts are ap- and TVCV has been identified in samples of dried leaf ple, European pear, Japanese pear, Japanese apricot, cit- material of A. chinensis from both China and Italy (Co- rus and lilies, and experimentally it infects more than 40 hen et al., unpublished information). Both viruses were species in 17 plant families (Yoshikawa, 2000). It is amplified by the primers designed to detect RMV (Cha- probably found wherever apples are grown and natural van et al., 2012) and can only be distinguished by se- spread has also been reported in citrus in China and 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 227

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Japan (Yoshikawa, 2000). Some Lilium ASGV strains 1979; Zhang et al., 1988; Yoshikawa, 2000). can infect Citrus, and a Pyrus isolate infects Citrus For diagnostic purposes ASGV was successfully de- (Yoshikawa, 2000). The kiwifruit ASGV isolate from A. tected in infected indicator plants and directly from Ac- chinensis (AF522459) (Clover et al., 2003) has an identi- tinidia samples by conventional RT-PCR using the cal genomic organization to strains from Citrus, Malus primers (ML-F and ML-R, Table 2) of Ito et al. (2002). and Lilium, with a high degree of identity to Citrus ASGV was also detected by ELISA, using ASGV antis- (D16681), Malus (D14995) and Lilium (AB004063) iso- era raised against apple strains of ASGV (Table 2), and lates across the 32-terminal half (2,901 nt) of the ICRT-PCR. Both protocols were reliable but the ICRT- genome. The coat protein and movement protein genes PCR was 50 times more sensitive than ELISA (Clover et share a nucleotide identity of >95% with other strains al., 2003). Because the ASGV is thought to be transmit- of ASGV (Clover et al., 2003). The morphological, epi- ted in the field only by grafting, planting virus-free demiological, serological and molecular characteristics plants is the best means of controlling the virus. ASGV of the virus from A. chinensis are indistinguishable from does not represent a threat to kiwifruit production. those of ASGV from other hosts (Clover et al., 2003). ASGV in kiwifruit was first detected in A. chinensis Cucumber necrosis virus. CNV (genus Tombusvirus, budwood from Shaanxi province, (China), grafted onto family Tombusviridae) is an isometric virus of 31 nm di- healthy rootstocks of A. chinensis cv. Hort16A and ameter containing ssRNA (Dias, 1972). CNV was first grown in post-entry quarantine in New Zealand. The described in 1959 on cucumber plants from Canada original source of the plants, within China, is not which appeared stunted with severe foliar symptoms known. Infected plants developed interveinal mottling, (McKeen, 1959). The virus is transmitted in soil by chlorotic mosaics and ringspots (Clover et al., 2003). zoospores of the fungus Olpidium radicale [syn. O. bor- However, these plants were subsequently found to be novanus, O. cucurbitacearum; (Dias, 1970a, 1970b)] but co-infected with RMV and vitiviruses (R.R. Chavan, un- not through seeds (McKeen, 1959). CNV can be me- published information). ASGV is often latent in com- chanically transmitted to a wide host range including mercial Malus and Citrus although it can cause graft plants belonging to the families Amaranthaceae, Aster- union necrosis, tree decline and death in some apple aceae, Chenopodiaceae, Cucurbitaceae, Fabaceae and (Yanase, 1983) and citrus (Broadbent et al., 1994) root- (Dias, 1972). However, to date, the virus has stock/scion combinations. It is unknown whether only been found naturally to infect cucumbers (Cucumis ASGV results in significant yield losses in A. chinensis sativus) in Canada (McKeen, 1959), lettuce (Lactuca as it was detected in plants detained in post-entry quar- sativa) and tomato (Solanum lycopersicum) in the USA antine under greenhouse conditions and observed for (Obermeier et al., 2001), and kiwifruit (Actinidia spp.) only a limited period of time (Clover et al., 2003). Some in China, Italy and New Zealand (Lebas et al., unpub- surveys for ASGV in A. chinensis have been carried out lished information). in New Zealand and ASGV was detected in extracts In 2009, A. arguta and A. deliciosa plants were from some plants using RT-PCR and immunocapture- bought from a commercial garden centre in Auckland RT-PCR (ICRT-PCR). Sequencing of amplicons con- (New Zealand) to be used as healthy controls for PCR. firmed the presence of ASGV, but repeated extractions Both plants were found to be infected with CNV when from the same plants gave variable results, indicating tested by ICRT nested-PCR (Table 2). The 215 bp se- that the virus was unevenly distributed in the plants. At- quences obtained from both species were identical tempts to isolate ASGV from orchard plants by inocula- (KC478972, KC478973) and had 99% nucleotide iden- tion to herbaceous indicator plants have never been suc- tity with CNV isolates from Canada (M25270) and cessful (Cohen et al., unpublished information). New Zealand (DQ663769). Subsequent testing of im- ASGV is transmissible by grafting and mechanical in- ported Chinese A. deliciosa (KC478971) and Italian A. oculation to herbaceous plants. Vectors and natural deliciosa plants confirmed the presence of CNV in this means of field transmission are unknown for isolates material (B.S.M. Lebas, unpublished information). Ac- from Actinidia, Malus or Citrus (Yoshikawa, 2000; tinidia arguta and A. deliciosa plants were propagated in Clover et al., 2003). ASGV is seed-transmitted in Lilium a local nursery that provides plants to commercial gar- longiflorum and C. quinoa (Inouye et al., 1979) but it is den centres all around New Zealand, so CNV is likely to unknown whether the Actinidia isolates are seed-trans- be widely distributed within the country. missible. The Actinidia isolate was graft-transmitted to CNV causes necrotic spots, severe leaf distortion and A. deliciosa and produced the same symptoms as in the stunting on greenhouse cucumber plants (McKeen, original host. It was also mechanically transmissible to a 1959). It elicits localized leaf necrosis on lettuce and number of herbaceous hosts (Clover et al., 2003). The was found in mixed infection with Lettuce necrotic symptoms observed on C. quinoa, Phaseolus vulgaris stunt virus (LNSV, tenative species in the genus and Vigna unguiculata are very similar to those de- Tombusvirus) on tomato with leaf chlorosis and internal scribed for isolates from other hosts (Inouye et al., fruit necrosis in the USA (Obermeier et al., 2001). No 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 228

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symptoms were observed on the two infected Actinidia the past 7 years. After purification of AVX from N. occi- plants from New Zealand or on the imported material dentalis, an antiserum was prepared from rabbit. Its from China and Italy. In addition, CNV was only de- successful use in indirect ELISA (plate-trapped antigen tected by ICRT nested-PCR, suggesting it was present ELISA) was demonstrated from infected herbaceous in- at a very low titre in all the Actinidia spp. plants tested. dicators and leaves of A. chinensis seedlings that had Therefore, it is likely that CNV is not a major pathogen been inoculated with the virus. AVX was detected at of kiwifruit. Although CNV is detected in an increasing high titre in inoculated leaves of A. chinensis seedlings, number of hosts, it has not been reported to cause any but its titre gradually declined in new leaves over several significant economic damage since the first report in months (Pearson et al., 2011). Inoculated leaves on 1959 (McKeen, 1959). CNV may have been present in these seedlings showed veinal necrosis but no symptoms New Zealand for some time. However, it has not been were observed on systemically infected leaves (D. Co- reported on any other crop species, although the vector hen and A.G. Blouin, unpublished information). AVX O. radicale infects cucumber, tomato and beans (Penny- can also be detected by RT-PCR (Table 2). This virus cook, 1989). The impact of CNV on the kiwifruit pro- has so far only been isolated from Actinidia spp on to duction is unknown but is likely to be negligible. Nicotiana spp and C. quinoa no further information is available on its host range and distribution. However, Actinidia virus X. AVX is a novel putative potexvirus based on the absence of symptoms in systemically in- isolated on herbaceous indicator plants from three A. fected A. chinensis seedlings and the low incidence of chinensis plants. The virus has flexuous particles of detection, the impact of AVX is likely to be very low. about 485 nm long and 12-13 nm width. Its sequence (KC568202) shows the typical organisation of a po- texvirus with five ORFs. ORF1 (nt 26-4825) encodes KIWIFRUIT-ADAPTED VIRUSES the putative replicase of 1,599 aa with a calculated mass of 180 kDa. It contains the methyltransferase domain at Actinidia citrivirus. The Actinidia citrivirus has a the N-terminal, the NTPase/helicase domain in the cen- monopartite, linear, positive-sense, ssRNA genome of tral region and the RNA-dependant RNA-polymerase 8782 nt (JN900477) and shares 74% nucleotide identity domain in the C-terminal region (Martelli et al., 2007). with CLBV (AJ318061). The genome organisation is ORF1 is followed by a short intergenic region of 52 nt identical to that of CLBV, with three non-overlapping and the triple gene block (TGB) formed by three over- open reading frames and a 3’ terminus poly(A) tract. lapping ORFs; ORF2 (nt 4878-5585), ORF3 (nt 5554- ORF1 (nt 72-6035), the putative replicase polyprotein, 5916) and ORF4 (nt 5753-6022) have a calculated mass includes methyltransferase, AlkB, OTu-like peptidase, of 26, 13 and 10 kDa respectively. ORF5 (nt 6041-6784) papainlike protease, RNA helicase, and RNA-depend- codes for a 26 kDa coat protein. Phylogenetic analysis ent RNA polymerase domains, typical of a citrivirus showed the virus clustered with a subgroup comprising (Martelli et al., 2007). It codes for 1987 aa and has a cal- Narcissus mosaic virus (NMV), Asparagus virus-3 (AV-3), culated mass of 230 kDa. ORF2 (nt 6035-7123) codes Malva mosaic virus X (MaMV) and Scallion virus X for a putative movement protein of 362 aa has a calcu- (ScVX). The nucleotide identity on the full genome var- lated mass of 40 kDa. An intergenic region of 55 nts fol- ied between 64 and 65% with these viruses, and be- lows ORF2 before the start codon of ORF3 (nt 7124- tween 57 and 59% nt identity with Alstroemeria virus X 7178). ORF3 codes for a 40 kDa coat protein (358 aa). (AlsVX), Lettuce virus X (LVX) and Pepino mosaic virus The 5’ and 3’ UTRs are 71 and 526 nt long, respectively (PepMV). AVX was easily mechanically transmissible to (Chavan et al., 2013). CLBV is the type and currently N. benthamiana, N. clevelandii, and N. occidentalis, and the only recognised member of the genus Citrivirus. it induced systemic symptoms in C. quinoa. The Actinidia citrivirus has been detected only in ki- Two out of the three isolations of the virus were wifruit scionwood material imported from China (Cha- made from samples of symptomatic kiwifruit. In these van et al., 2013). In A. chinensis the virus is associated two plants, a vitivirus was also detected. The two symp- with a range of symptoms, including vein clearing and tomatic plants were destroyed after sample collection mild mottling on leaves and interveinal chlorosis during and resampling was not possible. The third detection summer, although some infected accessions remained was from a symptomless plant but re-isolation, RT-PCR symptomless. All of the symptomatic kiwifruit plants in- and ELISA failed to re-detect the virus. It is possible fected with the Actinidia citrivirus were found to be co- that the virus is cryptic in kiwifruit in the same way that infected, making if difficult to attribute the symptoms AlsVX is latent in Alstroemeria (Fuji et al., 2005). Ki- to one virus alone (Fig. 2B shows leaf symptoms of a wifruit may not be the preferred host of AVX. The virus plant co-infected with Actinidia citrivirus, AcVA and is probably distributed unevenly in kiwifruit plants and AcVB). may occur at low titre, as it was only isolated on three No attempt has been made to inoculate the Actinidia occasions out of many hundreds of inoculations over isolate to citrus, the only known natural host of CLBV. 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 229

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The Actinidia citrivirus is transmitted by grafting in Ac- Most vitiviruses naturally infect a single host; the other tinidia, similarly to CLBV (Vives et al., 2001). CLBV is natural vitivirus hosts currently known are mint (Mint also transmitted by contaminated knife blades (Rois- virus 2, MV2) and heracleum (Heracleum latent virus, tacher et al., 1980) and at a low percentage through HLV) (Adams et al., 2011). seeds (Guerri et al., 2004), but so far there is no evi- Two novel vitiviruses Actinidia virus A (AcVA) and dence that the Actinidia citrivirus can be mechanically Actinidia virus B (AcVB) were detected in kiwifruit by transmitted by orchard operations and no seed trans- RT-PCR (Blouin et al., 2012). Both viruses have a mission was observed within more than 300 Actinidia monopartite, linear, positive-sense, ssRNA genome. seedlings of an infected A. chinensis female parent; sug- AcVB genome was fully sequenced (JN427015) and is gesting that if there is any seed transmission in kiwifruit, 7488 nt long and 7566 nt of AcVA were sequenced it would be at very low rate (D. Cohen and A.G. Blouin, (JN427014) covering all the genome but the 5’UTR and unpublished information). The Actinidia citrivirus and the beginning of the ORF1. They share 64% nucleotide CLBV have both been mechanically transmitted to a identity and each comprises five ORFs: ORF1 codes for range of common herbaceous indicator plants including the replication genes with a calculated mass of 195 kDa. N. benthamiana, N. clevelandii, N. glutinosa and N. occi- Both sequences include conserved domain for a methyl- dentalis; the citrus isolate of CLBV gave symptomless transferase, an AlkB, a RNA helicase and a RNA-de- infections (Vives et al., 2008; Guardo et al., 2009) pendent RNA-polymerase in respective order from the whereas the Actinidia isolate produced distinctive symp- amino terminus to the carboxyl terminus as described toms on N. glutinosa (Fig. 2C) (Chavan et al., 2013). for the genus in Martelli et al. (2007); AcVA has a ly- Although Actinidia citrivirus isolates can be detected sine-rich insert between motifs I and II of the methyl- by ELISA using an antiserum against Dweet mottle transferase that is not present in other vitiviruses, in- virus [= CLBV (Antiserum USDA253, courtesy of Dr. cluding AcVB. ORF2 codes for a putative protein of Richard Lee] (D. Cohen and A.G. Blouin, unpublished unknown function and has a calculated mass of 25 and information), and by PCR using primers designed from 27 kDa for AcVA and AcVB respectively. This is the the coat protein gene of CLBV (Table 2), the Actinidia most divergent gene of the virus with only 16% aa iden- citrivirus shows several distinct differences. First, the tity between them and no homology to any protein from symptoms induced in N. glutinosa (Fig. 2C). Second, all GenBank; ORF3 (nt 5704-6597 and 5698-6570) codes sequences of CLBV deposited in GenBank show very for a movement protein with a calculated mass of 33 high similarity with one another, whereas the Actinidia and 32 kDa respectively, and share 56% aa similarity. citrivus isolates show considerable sequence variation. ORF4 (nt 6515-7111 and 6488-7084) codes for the coat Third, phylogenetic analysis has shown that from the 3’ protein of a calculated mass of 21 kDa for both viruses. end of ORF1 to the 3’ untranslated region (UTR) (in- This is the most conserved gene of the viruses and Ac- cluding all of ORF2 and ORF3) the citrus CLBV and VA and AcVB share 75% aa in common and are less the Actinidia citrivirus share 78% identity at the nt level than 70% aa similar to the closest vitiviruses (GVB and and > 90% identity at the aa level. However, the 5’ and HLV). ORF5 (nt 7112-7429 and 7085-7405) codes for a 3’ UTRs, as well as the 5’ end of ORF1, show diver- putative RNA binding (RNA silencing inhibitor) pro- gence of about 30% at the nt level (Chavan et al., 2013). tein of a calculated mass of 12 kDa (Blouin et al., 2012). Based on current International Committee on Taxono- As a consequence of the historical movement of plant my of Virus (ICTV) demarcation criteria for sequence material, the grapevine-infecting vitiviruses have been similarity within the family Betaflexiviridae, i.e. less than reported in most grapevine-growing regions. Vitiviruses 72% nt identity or 80% aa identity in the CP or the are not known to be seed-transmitted and AcVA and polymerase gene (Adams et al., 2011), Actinidia cit- AcVB have only been detected in accessions that were rivirus is borderline for classification as a new species. imported to New Zealand as scions, or in scions that Since means of natural spread of the Actinidia cit- have been grafted on to an infected plant (Blouin et al., rivirus are unknown, control relies on the use of virus- 2012). AcVA and AcVB have also been detected in two free scionwood and rootstocks in combination with Chinese scionwood accessions growing in Italy (D. Co- good hygiene to prevent the possibility of mechanical hen and A.G. Blouin, unpublished information). transmission via pruning. The impact of the virus is like- Inoculation of sap from symptomatic vines of A. chi- ly to be very low, mostly due to the lack of a vector. nensis induced symptoms on N. occidentalis. The coat protein was partially purified from herbaceous indicator Actinidia virus A and Actinidia virus B. The genus plants and a few peptides common to GVB were identi- Vitivirus was named after Vitis sp., host of the reference fied by tandem mass spectrometry (Blouin et al., 2010). species Grapevine virus A (GVA). Vitis vitifera also A survey of more material showed symptoms ranged hosts four additional vitiviruses, i.e. Grapevine virus B, from large ringspots, vein chlorosis and mottle to symp- Grapevine virus D, Grapevine virus E and Grapevine tomless plants, but some of the infected plants could virus F (Adams et al., 2011; Al Rwahnih et al., 2012). host more viruses (Fig. 2B showing symptoms from a 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 230

230 Viruses of kiwifruit Journal of Plant Pathology (2013), 95 (2), 221-235

mixed infection including AcVA, AcVB and the Actini- Secoviridae (Sanfaçon et al., 2012). CLRV has been re- dia citrivirus). AcVA and AcVB were transmitted by ported to be present in North America, Chile, Peru, Eu- grafting to A. deliciosa but the infected plants remained rope, China, Japan, Australia and New Zealand (Woo et mostly symptomless (Blouin et al., 2012). al., 2012a). In addition to its worldwide distribution, Grapevine vitiviruses are spread by mealybugs and the virus also has a wide natural and experimental host scale insects. Vitiviruses are often detected as coinfec- range, infecting members of more than 36 plant families tions with a member from the family Closteroviridae. In (Walkey et al., 1973; Rebenstorf et al., 2006). This in- grapevine, Grapevine leafroll associated virus 1 (GLRaV- cludes a variety of wild and cultivated, herbaceous and 1 genus Ampelovirus) has been reported to be co-trans- woody plant species. Unlike most nepoviruses, CLRV mitted with the GVA (Hommay et al., 2008) . On some does not appear to be transmitted by soil-inhabiting ne- occasions, the vitivirus can be transmitted alone. A re- matodes. However, the virus has been documented to cent study using a donor plant with mixed infection of be transmitted by seed, pollen, grafting and mechanical GVA and Grapevine leafroll associated virus 3 (GLRaV- inoculation to herbaceous hosts (Woo et al., 2012a). 3) found that the majority of the receiving plants were CLRV has a bipartite genome of two positive-sense, ss- infected with GLRaV-3 alone (24%) or both viruses RNA molecules. Each RNA molecule is encapsidated (31%), while only 2% were infected with GVA alone separately in an isometric particle that is about 28 nm in and 43% were not infected (Blaisdell et al., 2012). diameter. Both RNA molecules are required for virus in- In kiwifruit, no movement of the Actinidia vitiviruses fection (Le Gall et al., 2005). RNA-1 and RNA-2 have has been observed in New Zealand other than by graft- structural organization typical of the genus and comprise ing and all the positive vines could be linked to an im- 7905 and 6511 nt, respectively (Eastwell et al., 2012). port of scionwood from China (Blouin et al., 2012). CLRV was first described in sweet cherry in England Some of the plants had been imported for several (Posnette and Cropley, 1955). Subsequently, it was decades. This lack of movement suggests that the virus found to cause leaf rolling and plant death in cherry is present either without its helper virus or without effi- (Cropley, 1961) and a range of other plant species in- cient vectors. All the novel vitiviruses were detected in cluding elderberry, olive, raspberry, rhubarb, walnut co-infection. It is possible that both viruses share a com- and a number of other shrub, tree, weed and ornamen- mon vector (before the introduction to New Zealand), tal species (Büttner et al., 2011; Woo et al., 2012a). resulting in co-infection; however, it is expected that CLRV was isolated from a A. chinensis cv. Hort16A or- both viruses may also exist as single infections in the chard in which vines were showing necrotic symptoms wild. on leaves (Fig. 2D), as well as cane die-back and bark A virus that may potentially assist the natural trans- cracking. Some of the fruit from the infected vines do mission of Actinidia vitiviruses was identified by next not have the beak at the calyx end that is characteristic generation sequencing (NGS) from a consignment of ki- of the Hort16A cultivar (Fig. 2E). Additionally, the fruit wifruit imported from China and held in quarantine in from infected vines are uneven in size, and the crop New Zealand. This virus has the characteristics of a yield is reduced. Extracts from symptomatic leaves in- member of the family Closteroviridae, but the sequence oculated to herbaceous indicators induced large necrot- analysis shows that it is distant from any characterised ic lesions on N. occidentalis and ringspots on N. species of this family (Blouin et al., unpublished infor- tabacum. The virus was identified by RT-PCR and se- mation). Based on the heat shock protein 70 (HSP70), quencing. The sequences obtained from infected ki- the most conserved gene within the family Closteroviri- wifruit (JN371141) closely match those of an isolate dae, the closest relative (37% amino acid identity) was from raspberry in New Zealand (Jones and Wood, Olive leaf yellowing associated virus (OLYaV) 1978), and described as group C (Rebenstorf et al., (AJ440010), an unclassified member of this family. Fur- 2006). Detection in symptomatic material is also possi- ther characterisation of this novel virus, including full ble with DAS-ELISA (Table 2). CLRV was also detect- sequence and transmission studies, may clarify its possi- ed in Rumex spp. (JN371148) directly below the infect- ble role as a helper virus. ed vines using DAS-ELISA. A mechanism for the move- The impact of vitiviruses on kiwifruit largely depends ment of the virus between different hosts has not yet on their capacity to move and is therefore low in New been identified. Within kiwifruit, the virus seems to Zealand. It is also too early to assess the impact of the spread along the row, suggesting a possible mechanical novel putative closterovirus. spread by pruning/girdling equipment. All these charac- teristics make CLRV a potential threat for kiwifruit pro- duction and future studies are required to understand DISEASE-INDUCING VIRUSES fully its ecology.

Cherry leaf roll virus. CLRV is an established Pelargonium zonate spot virus. PZSV is the type species within subgroup C of genus Nepovirus, family species and the single member of the Anulavirus genus 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 231

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within the Bromoviridae family (Bujarski et al., 2012). Poor data are available on the variability within PZSV Amazon lily mild mottle virus, a new virus, isolated isolates. High amino acid identity has been reported be- from an Amazon lily plant, has been recently described tween Italian and Israeli tomato isolates (93% ORF 1a, and proposed as new anulavirus species (Fuji et al., 97% ORF 2a, 98% ORF 3a and 96% ORF 3b) (Lapidot 2012). PZSV was described as Tobacco streak virus et al., 2010). Similar results have been obtained compar- when first detected on tomato plants in southern Italy ing the Italian isolates from tomato and kiwifruit (92, 99, (Martelli and Cirulli, 1969) and later designated as 98 and 100% aa identity, respectively). PZSV when isolated from Pelargonium zonale PZSV induces conspicuous concentric chrome-yel- (Quacquarelli and Gallitelli, 1979). This virus has been low bands in the leaves of P. zonale, from which its reported on tomato, pepper and weed species from name is derived, and is the causal agent of a severe Italy, Spain, France, the USA, Israel and Australia (Gal- tomato disease characterized by concentric chlorotic/ litelli, 1982; Luis-Arteaga and Cambra, 2000; Gebre-Se- necrotic rings and line patterns of leaf stems and fruits lassie et al., 2002; Liu and Sears, 2007; Escriu et al., together with plant stunting, leaf malformation, and re- 2009; Lapidot et al., 2010; Luo et al., 2010). As well as duced fruit set, which often result in plant death (Gal- tomato, pepper and geranium, PZSV also naturally in- litelli, 1982). Data from preliminary studies on PZSV-in- fects Cynara cardunculus var. scolymus (globe artichoke), fected kiwifruit plants suggest that the virus decreases Capsella bursa-pastoris, Chrysanthemum segetum, vigour year by year and then productivity of the plants. Diplotaxis erucoides, Picris echioides, Sonchus oleraceus, Moreover, infected fruit exhibit progressive decreased Cakile maritima. PZSV has been transmitted to herba- metabolic activity and significant reduction of cell wall ceous plants in 29 species, within nine dicotyledonous water content, indicating early senescence of tissues in families, by mechanical inoculation (Martelli and Cirul- PZSV-infected fruit compared with uninfected samples. li, 1969; Gallitelli et al., 1983). The virus can be successfully transmitted from A. chi- Recently, PZSV has been detected in several sympto- nensis to indicator plants, including C. quinoa, N. ben- matic kiwifruit plants (A. chinensis cv. Hort16A) in thamiana, N. glutinosa and N. tabacum, by mechanical Italy, from two orchards located in the Emilia-Romagna inoculation during spring but efficiency decreases dur- region. Infected plants showed chlorotic and necrotic ing summer or autumn. rings on leaves (Fig. 2F) and depressed areas on the PZSV can be detected directly from symptomatic ki- fruits that resulted in deformation of the berries (Bic- wifruit tissues by ELISA, dot blot DNA hybridization cheri et al., 2012). Four infected plants were identified and RT-PCR (Table 2), and in symptomless plants by during 2011 and three additional plants were identified RT-PCR. in 2012. Symptoms appeared early in the spring and re- PZSV is seed-borne in Diplotaxis erucoides and N. mained evident until the end of the season in plants glutinosa. The virus is associated with the pollen and with severe infection but disappeared at the beginning transmitted by thrips feeding on flowers of susceptible of summer in plants with mild or sectorial infection. hosts (Vovlas et al., 1989; Gallitelli et al., 2005). In Moreover, cuttings obtained from symptomatic plants tomato, PZSV is transmitted by seed, with efficiency of developed infected but symptomless leaves suggesting 29%, and by pollen, although infected pollen cannot that a long incubation period, and therefore high viral transmit the virus to mother plants, only to the seed titre, may be necessary for symptom expression. (Lapidot et al., 2010). Particles of PSZV are non-enveloped and quasi- No data are available about transmission from herba- spherical, with a diameter of 25-35 nm, and the coat ceous host to kiwifruit and whether transmission occurs protein is about 23 kDa (Gallitelli et al., 2005). The se- naturally between kiwifruit plants. New studies are quence of the complete genome has been obtained from therefore necessary to better investigate the biological the Italian tomato isolate; it is divided into three RNA and molecular proprieties of PZSV that infect kiwifruit species encoding four proteins (Finetti-Sialer and Gal- and its role as a causal agent of disease in Actinidia sp. litelli, 2003). RNA-1 is 3383 nt long, with a single ORF With regard to the symptoms in the commercial or- encoding a polypeptide which contains conserved mo- chard, PZSV is an important pathogen to manage. Fur- tifs of type I methyltransferases and of the helicases of ther study will assess its spread efficiency, which will de- superfamily 1. RNA-2 is 2435 nt long and encodes a termine the seriousness of the disease. polypeptide (ORF2) showing identity to the RNA-de- pendent RNA polymerases of positive-strand RNA viruses. RNA-3 is 2659 nt long and contains two ORFs. The product of ORF3a shows similarities with the 30K CONCLUSION superfamily of virus movement proteins and ORF3b en- codes the viral coat protein, which is expressed via the Worldwide, the kiwifruit industry is relatively new subgenomic RNA-4 (Finetti-Sialer and Gallitelli, 2003; and is based on mainly two species: A. deliciosa, repre- Gallitelli et al., 2005). senting the vast majority of the commercial production; 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 232

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and A. chinensis, which comprises most of the newest they are being monitored. cultivars. Cultivars of these two species are among the Kiwifruit virus research was initiated following the first from a recently domesticated plant family which identification of ASGV during quarantine surveillance. holds promise for many further new commercial culti- Subsequent research identified the presence of Actini- vars (Ferguson and Seal, 2008). Likewise, research on dia citrivirus, AcVA, AcVB, RMV and CNV in the same kiwifruit viruses is in its infancy. The viruses listed in consignment of plant material (Chavan et al., unpub- this review were identified from only four laboratories, lished information). The rigorous quarantine system in three in New Zealand and one in Italy, and all have been place in New Zealand has therefore demonstrated its identified in kiwifruit over the past decade. The in- importance. This review compiles a list of diagnostic creased interest in disease-resistant cultivars of kiwifruit tools that are now available to researchers and research as well as the recent discovery of pathogenic viruses laboratories as well as quarantine facilities for the cur- should stimulate further research. New technologies rent list of viruses known to infect kiwifruit. Table 2 in- such as NGS will probably identify many new viruses, dicates that RT-PCR is the most sensitive and reliable especially since this method does not require prior method for detection of these viruses in kiwifruit, al- knowledge about infecting viruses. Such technologies though ELISA has also been widely used as a routine will be useful to identify more RNA and DNA viruses detection method. and/or viroids, and to pinpoint rapidly the cause of dis- The identification of these 13 viruses that can infect ease when present, but could also uncover latent virus- kiwifruit has important repercussions for orchard man- es, and potentially viruses that are beneficial (Roossinck, agement, especially for nurseries that propagate ki- 2011). A major challenge is to undertake the basic re- wifruit. It is important to have nucleus stock plants that search on the virus ecology so that vectors, host range are free of known viruses. These health precautions and impacts on the plant host can be characterised. should preclude the chance of infection from the spe- We describe here 13 viruses that have been isolated cialist group of viruses. However, for PZSV and CLRV from kiwifruit. Many of these viruses are not associated there is a potential for transmission from reservoir hosts with important symptoms and/or spread (the non-spe- to kiwifruit and subsequent spread by pollen or me- cialists) and are not considered to be economically im- chanical transmission that needs to be better investigat- portant in commercial orchards. Of the 13 viruses pre- ed. Infected plants should be removed and equipment sented in this review, five (AVX, Actinidia citrivirus, Ac- should be cleaned after use on infected vines. These VA, AcVB and a novel putative closterovirus), to date, virus infections are of sufficient importance that infec- have not been isolated from another host. With the ex- tions should be confirmed by local diagnostic laborato- ception of AVX, these are putatively kiwifruit specialists, ries and/or reported to local phytosanitary agents to de- as they are related to viruses that have a narrow host termine whether further actions are required. range. These kiwifruit-specialist viruses mostly cause leaf Kiwifruit breeding has a remarkable depth of genetic symptoms, but can also be latent. However, since these variation to exploit for new commercial attributes such viruses have only been studied in non-commercial or- as flavours, colours and nutritional benefits (Ferguson chards, their effect on yield and plant longevity is un- and Huang, 2007). The germplasm should also be known. No vector of these viruses has been identified yet screened for those vines that are either resistant or toler- and no movement to new kiwifruit plants has been ob- ant to each of these 13 viruses. The range of symptoms served except by grafting. Since it is likely that these observed to date for some individual viruses on differ- viruses originate from wild kiwifruit populations in Asia, ent cultivars suggests that there is potential for such insect vectors are probably present in these countries. virus resistance or tolerance. To date the industry has These specialist (or host adapted) viruses can infect been fortunate to have selected a cultivar, A. deliciosa plants without symptoms and would be easily overlooked cv. Hayward, that has shown very good resistance or tol- and propagated within nurseries or orchards. The spe- erance to disease in general and viruses in particular. cialist viruses might pose a risk to kiwifruit growing with- Currently there is a need for tolerance to the bacterium in new environments if they infect new cultivars, interact P. syringae pv. actinidiae but screening for virus toler- with other viruses, or if a vector is present. ance would also be prudent. This review provides a The third group of kiwifruit-infecting viruses at pres- starting point for further studies to screen, identify and ent comprises CLRV and PZSV, two viruses that pose research viruses and plant-virus interactions in kiwifruit. more serious threats with respect to symptoms and spread. These two viruses are pollen-borne and seed- transmitted, although it is not yet known if this occurs ACKNOWLEDGEMENTS in kiwifruit. Although the two viruses were identified recently, they have already been associated with signifi- The authors would like to thank Ross Ferguson, Mark cant symptoms, with consequences for yield. It is too Andersen and Kieren Arthur for critical reading of this early to assess the spread of these virus infections, but manuscript and for providing valuable comments. We are 001_JPP_Review_221_colore 30-07-2013 16:52 Pagina 233

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