Viroid Prevalence in Tunisian Citrus

The citrus industry in Tunisia is based mainly on the production of local cultivars of sweet orange (Citrus sinensis), common mandarin (C. reticulata), clementine (C. clementina), and lemon (C. limon). Sour orange (C. aurantium) is the only rootstock presently being used in the major growing area located at Cap Bon where 80% of citrus is being produced. The presence of tristeza disease in the Mediterranean basin is a threat to the Tunisian citrus industry, and new rootstocks giving tristeza tolerant rootstock/scion combinations are urgently needed as an alternative to sour orange. Since some promising rootstocks are known to be sensitive to viroids (1), a survey was conducted to determine if the cultivars grown presently in Tunisia were infected with viroids. Following a preliminary report (2), an extensive survey was conducted from 1995-2001 that included 174 symptomless sources being grown at Cap Bon: 26 Maltaise demi-sanguine, 9 Maltaise sanguine, 20 Maltaise blonde, 4 orange doublefine, 16 Washington navel, 12 Valencia late, 29 common mandarin, 42 Cassar clementine, 5 Lunari lemon, and 11 Eureka lemon. These sources were graft-inoculated into Etrog citron that subsequently developed symptoms characteristic of viroid infection. Sequential polyacrylamide gel electrophoresis analysis and molecular hybridization using viroid-specific probes (1) revealed that all sources were infected with at least two viroids. Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), and Citrus viroid III (CVd-III) were widespread and accounted for 68.4, 67.8, and 81.0% of the sources tested, respectively. Citrus bent leaf viroid (CBLVd) and Citrus viroid IV (CVd-IV) were only found in 32.7 and 2.3% of the sources. The most frequent viroid combinations were CEVd+HSVd+CVd-III (17.8%) and CEVd+CVd-III (17,2%), whereas HSVd+CVd-IV and CEVd+CBLVd+CVd-III+CVd-IV were found in a single source (0.6%). References: (1) N. Duran-Vila and J. S. Semancik. Pages 178–194 in: Viroids. CSIRO Publishing, Australia, 2003. (2) A. Najar et al. Pages 398–400 in: Proc. 15th Conf. Int. Org. Citrus Virol, 2002.

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A Survey of Citrus Viroids in Campania (Southern Italy)

Plant Disease ◽

10.1094/pd-89-0434c ◽

2005 ◽

Vol 89 (4) ◽

pp. 434-434

Author(s):

M. Malfitano ◽

M. Barone ◽

D. Alioto ◽

N. Duran-Vila

Keyword(s):

Southern Italy ◽

Sweet Orange ◽

Poncirus Trifoliata ◽

Sour Orange ◽

Campania Region ◽

First Report ◽

Hop Stunt Viroid ◽

Washington Navel ◽

Viroid Infection ◽

Citrus Viroids

Within a certification program of citrus, a survey covering 14 citrus fields not previously screened were randomly selected in seven areas of the Campania Region of southern Italy to evaluate the presence and distribution of citrus viroids in the region. During the last 3 years, 112 trees were visually inspected and sampled including 14 ‘Navelina’, 8 ‘Washington navel’, 13 ‘Biondo Commune’, 10 ‘Tarocco’, 6 ‘Valencia late’, 1 ‘Sanguinello’, and 1 ‘Vanilla’ sweet oranges (Citrus sinensis), four sour oranges (C. aurantium), 21 ‘Commune’ and 7 ‘Monreale’ clementines (C. clementina), 8 ‘Common’ and 2 ‘Avana’ mandarins (C. reticulata), 8 ‘Ovale di Sorrento’ and 6 ‘Zagara’ lemons (C. lemon), 2 Tangelos (C. reticulata × C. paradisi), and 1 grapefruit (C. paradisi). Nearly all (37 of 38) mandarin and clementine trees showed cachexia-like symptoms. Severe bark scaling that is characteristic of exocortis disease was observed on a single ‘Ovale di Sorrento’ lemon, the only tree incidentally found to be grafted onto the exocortis-sensitive trifoliate orange (Poncirus trifoliata) rootstock, since sour orange (C. aurantium) is the main rootstock used in Campania. Samples (104 of 112) that had been graft-inoculated into Arizona 861-S1 ‘Etrog citron’ (C. medica L.) on rough lemon (C. jambhiri Lush.) rootstock showed typical viroid symptoms. Viroid infection was confirmed by sequential polyacrylamide gel electrophoresis analysis (2) and molecular hybridization of imprinted membranes (1). Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), and Citrus viroid III (CVd-III) were the most frequently detected, accounting respectively, for 67.9, 86.6, and 84.8% of the infected samples. Citrus viroid IV (CVd-IV) and Citrus bent leaf viroid (CBLVd) were found in 24.1 and 13.4% of the sources. CBLVd was detected in sweet orange (‘Tarocco’ and ‘Biondo Commune’) and lemon (‘Ovale di Sorrento’ and ‘Zagara’), while CVd-IV was detected in sweet orange (‘Tarocco’, ‘Biondo Commune’, and ‘Washington navel’), lemon (‘Ovale di Sorrento’ and ‘Zagara’), clementine (‘Commune’), and sour orange. Most trees (82.1%) were infected with more than one viroid species. The most frequent viroid combinations were CEVd + HSVd + CVd-III (40.2%), HSVd + CVd-III (16.1%), and CEVd + CBLVd + HSVd + CVd-III + CVd-IV (12.5%). To our knowledge, this is the first report of CEVd, CBLVd, HSVd, CVd-III, and CVd-IV in the Campania Region and the first report of CBLVd and CVd-IV in Italy. References: (1) A. Palacio et al. Eur. J. Plant Pathol.105:897, 2000. (2) R. F. Rivera-Bustamante et al. Anal. Biochem. 156:91, 1986.

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First Report of Hop stunt viroid in Apricot in China

Plant Disease ◽

10.1094/pd-90-0828c ◽

2006 ◽

Vol 90 (6) ◽

pp. 828-828 ◽

Cited By ~ 5

Author(s):

Y. A. Yang ◽

H. Q. Wang ◽

R. Guo ◽

Z. M. Cheng ◽

S. F. Li ◽

...

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Rt Pcr ◽

First Report ◽

Republic Of China ◽

Stunt Viroid ◽

Hop Stunt Viroid ◽

Viroid Infection ◽

Wide Range ◽

Irregular Border ◽

Apricot Tree

Hop stunt viroid (HSVd), a member of the family Pospiviroidae, was first described as the causal agent of hop stunt disease in Japan. It has since been found in a wide range of hosts including herbaceous and woody hosts (e.g., hop, cucumber, grapevine, citrus, plum, peach, pear, apricot, almond, and pomegranate). It was also detected and characterized in apricot where infection appears to be latent (1). The viroid occurs frequently in apricot. In southeastern Spain, the presence of HSVd was found to infect 81% of apricot trees (2). Apricots originated in China and are extensively cultivated, but HSVd infection in this host has not been reported. In September 2005, a single symptomatic apricot tree, ‘Yin Bai’, one of the most popular and widely grown cultivars in China, was discovered at the Institute of Fruit Science in Changping District in Beijing, Peoples Republic of China. Observed symptoms included a number of yellow spots with an irregular border that scattered in an irregular manner over the leaf surface. Total RNA was extracted and used for return-polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR) (4). Results of both assays were positive for HSVd. A 297-bp full-length DNA fragment was amplified by RT-PCR using primers R1 (5′-GCTGGATTCTGAGAAGAGTT-3′) complementary to HSVd residues 87–106 for the RT reaction, followed by R2 (5′-AACCCGGGGCTCCTTTCTCA-3′) complementary to HSVd residues 67–84 and forward primer F3 (5′-AACCCGGGGCAACTCTTCTC-3′) residues 79–96 for PCR. The primers are located in the strictly conserved central region of the conserved HSVd group and contain the unique endonuclease restriction site SmaI. The amplified products were cloned into pGEM-T (Promega, Madison, WI) and selected for further analysis on the basis of the results of restriction digests. Six individual clones were sequenced and three different sequences were obtained. Nucleic acid sequence (GenBank Accession No. DQ362901) obtained from one clone was 99.3% (nucleotide changes T206→C, C233→T) identical to HSVd.apr8 (GenBank Accession No. Y09349) (3). Sequence (GenBank Accession No. DQ362904) obtained from three clones was 99.7% (nucleotide change C233→T) and a third sequence (GenBank Accession No. DQ362905) obtained from two clones was 99.3% (nucleotide changes G107→A, C233→T) identical to HSVd.apr8. Further investigation is necessary to determine whether the symptoms observed are associated with the viroid infection. To our knowledge, this is the first report of HSVd isolated from apricot in China. References: (1) N. Astruc et al. Eur. J. Plant Pathol. 102:837, 1996. (2) M. C. Cañzres et al. Acta Hortic. 472:581, 1998. (3) S. A. Kofalvi et al. J. Gen. Virol. 78:3177, 1997. (4) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995.

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Citrus Viroids: Symptom Expression and Performance of Washington Navel Sweet Orange Trees Grafted on Carrizo Citrange

Plant Disease ◽

10.1094/pdis-05-14-0457-re ◽

2015 ◽

Vol 99 (1) ◽

pp. 125-136 ◽

Cited By ~ 12

Author(s):

N. Murcia ◽

S. M. Bani Hashemian ◽

P. Serra ◽

J. A. Pina ◽

N. Duran-Vila

Keyword(s):

Sweet Orange ◽

Fruit Production ◽

Symptom Expression ◽

Trifoliate Orange ◽

Carrizo Citrange ◽

Hop Stunt Viroid ◽

Washington Navel ◽

Canopy Volume ◽

Viroid Infection ◽

Orange Trees

Citrus are natural hosts of several viroid species. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are the causal agents of two well-known diseases of citrus, exocortis and cachexia. Other viroids have been found to induce specific symptoms and different degrees of stunting in trees grafted on trifoliate orange and trifoliate orange hybrids. A field assay was initiated in 1989 to establish the effect of CEVd, HSVd, Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) on Washington navel sweet orange trees grafted on Carrizo citrange rootstock. Here we report the effect of viroid infection on symptom expression, tree size, fruit production and quality evaluated from 2004 to 2007. Vegetative growth was affected by viroid infection with height and canopy volume being reduced. No bark scaling symptoms were observed in CEVd-infected trees albeit they presented lesions and blisters in the roots. Bark cracking symptoms were consistently observed in CBCVd-infected trees that were smaller with enhanced productivity and fruit size. No major effects were found as a result of infection with CBLVd, HSVd, or CDVd. The quality of the fruits was not affected by viroid infection, except for the low diameter of the fruits harvested from HSVd-infected trees. An interesting effect was identified in terms of tree productivity increase (yield/canopy volume) as a result of infection with CEVd, CDVd, and especially CBCVd.

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Detection of Multiple Infections of Citrus exocortis viroid, Citrus viroid III, and Hop stunt viroid Variants in Hunan Province, China

Plant Disease ◽

10.1094/pdis-91-9-1205a ◽

2007 ◽

Vol 91 (9) ◽

pp. 1205-1205 ◽

Cited By ~ 5

Author(s):

S. Rizza ◽

A. Catara ◽

X. F. Ma ◽

Z. Deng

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Leaf Roll ◽

Poncirus Trifoliata ◽

Hunan Province ◽

Variable Degree ◽

Rt Pcr ◽

Stunt Viroid ◽

Citrus Exocortis Viroid ◽

Hop Stunt Viroid ◽

Citrus Viroids

Citrus cultivation in China has increased since the late 1970s, with China now having the largest area of citrus in culture in the world that is spread in 22 provinces and municipalities. Hunan Province has undergone a program to become one of the major citrus producers in China. Poncirus trifoliata is the main rootstock, so citrus viroids are a limiting factor for further citriculture development. In mainland China, only the presence of Citrus exocortis viroid (CEVd) has been reported from Etrog citron indexing, sPAGE (sequential polyacrylamide gel electrophoresis) analysis (2), and reverse transcription (RT)-PCR (3). Three viroid-like RNAs, a1, b1, and d, based on sPAGE patterns were detected years ago in our laboratory in budsticks received from Sichuan Province. To identify different viroids and determine their distribution, a survey has been undertaken. Field trees showing stunting, bark scaling and cracking of the rootstock, and poor yield were tested using biological indexing and PCR for the most frequent citrus viroids. Samples from six trees of a local sweet orange variety and three of a Clementine variety introduced from abroad, both grafted on P. trifoliata and showing a variable degree of bark scaling and cracking, were collected near Changsha and in the County of Xin Ning at the end of summer 2006. Small pieces of bark were inserted in stems of young E. citron budwood grafted on rough lemon and maintained in a warm greenhouse (24 to 32°C). Indexing on E. citron showed mild epinasty and leaf roll typical of citrus viroid infections. To identify specific viroids, bark was ground to a fine powder with liquid nitrogen and total RNA was extracted with TRIZOL Reagent (Invitrogen, San Diego, CA) and tested by RT-PCR to detect CEVd, Hop Stunt viroid (HSVd), and Citrus viroid III (CVd-III), as well as to identify the cachexia variants of HSVd. Four primer pairs were used to test the RNA extracts by RT-PCR (1). All samples were infected by HSVd, eight with CVd-III, and six with CEVd. The cachexia variants of HSVd were detected in four of nine samples. Mixed infections were as follows: one sample had CEVd and HSVd, eight had HSVd and CVd-III, and five were infected by the three viroids. A second sampling 3 months after inoculation gave the same amplification patterns. The results show that at least three viroids are present in citrus orchards in Hunan Province. To our knowledge, this is the first report of cachexia variants of HSVd and CVd-III in China. The common occurrence of these viroids supports the need for proper indexing of mother trees and a specific shoot tip grafting program to create healthy budwood sources to provide healthy plants. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes, 20:105, 2006. (2) L. Han et al. Viroids. CSIRO Publishing, Melbourne, 283, 2003. (3). Q. Hu et al. Acta Bot. Sin. 39:613, 1997.

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First record of a Hop stunt viroid variant on Nagpur mandarin and Mosambi sweet orange trees on rough lemon and Rangpur lime rootstocks

Plant Pathology ◽

10.1111/j.1365-3059.2005.01194.x ◽

2005 ◽

Vol 54 (4) ◽

pp. 571-571 ◽

Cited By ~ 3

Author(s):

P. Ramachandran ◽

J. Agarwal ◽

A. Roy ◽

D. K. Ghosh ◽

D. R. Das ◽

...

Keyword(s):

Sweet Orange ◽

First Record ◽

Rangpur Lime ◽

Stunt Viroid ◽

Rough Lemon ◽

Hop Stunt Viroid ◽

Orange Trees

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TRACKING HOP STUNT VIROID INFECTION IN APRICOT TREES DURING A WHOLE YEAR BY NON-ISOTOPIC TISSUE PRINTING HYBRIDISATION

Acta Horticulturae ◽

10.17660/actahortic.2001.550.45 ◽

2001 ◽

pp. 315-320 ◽

Cited By ~ 8

Author(s):

K. Amari ◽

M.C. Cañizares ◽

V. Pallás ◽

A. Myrta ◽

S. Sabanadzovic ◽

...

Keyword(s):

Stunt Viroid ◽

Tissue Printing ◽

Hop Stunt Viroid ◽

Viroid Infection

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An important new apricot disease in Spain is associated with Hop stunt viroid infection

European Journal of Plant Pathology ◽

10.1007/s10658-007-9127-7 ◽

2007 ◽

Vol 118 (2) ◽

pp. 173-181 ◽

Cited By ~ 17

Author(s):

K. Amari ◽

D. Ruiz ◽

G. Gómez ◽

M. A. Sánchez-Pina ◽

V. Pallás ◽

...

Keyword(s):

Stunt Viroid ◽

Hop Stunt Viroid ◽

Viroid Infection

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First Report of Hop stunt viroid Infecting Japanese Plum, Cherry Plum, and Peach in Greece

Plant Disease ◽

10.1094/pdis-03-13-0235-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1662-1662 ◽

Cited By ~ 3

Author(s):

M. S. Kaponi ◽

P. E. Kyriakopoulou

Keyword(s):

Stone Fruit ◽

Economic Losses ◽

Prunus Domestica ◽

Rt Pcr ◽

Japanese Plum ◽

Stunt Viroid ◽

Hop Stunt Viroid ◽

Fruit Skin ◽

Viroid Infection ◽

Field Samples

Dapple plum and peach fruit is a widely distributed disorder of plum and peach resulting in significant economic losses (4). During a survey for the presence of Hop stunt viroid (HSVd) on stone fruit trees in Greece, samples from 30 European plums (Prunus domestica L., cvs. President, Tuleu Grass), 45 Japanese plums (Prunus salicina Lindl., cvs. Angeleno, Diamond, Santa Rosa), 12 cherry plums (Prunus domestica L. var. insititia (L.) Fiori & Paoletti of unknown cultivar), and 107 peaches (Prunus persica (L.) Batsch, cvs. Red Haven, Elberta, June Gold, Spring Crest, Lemonato) were collected in several orchards around Greece. Their fruit skin symptomatology indicated viroid infection (reddish dappling blotches and cracks in European and Japanese plum, green dappling in cherry plum, and light colored blotches and lines in peach). Samples were screened with tissue-print hybridization (TPH) for HSVd using a full length DIG-labelled riboprobe deriving from in vitro transcription of the positive control, a citrus isolate of HSVd (G. Vidalakis, CCPP, University of California, Riverside). In total, 44 out of the 194 trees surveyed were HSVd-positive with TPH. For a small number (40) of TPH-positive field samples, TNA phenol extraction from fruit skin, leaves, and bark and one-tube two-step reverse transcription (RT)-PCR assays followed, using a standardized protocol (3) with two different primer pairs, one new primer pair (this study) and a previously reported primer pair (2). RT-PCR analysis showed the presence of HSVd in peach and Japanese plum in prefectures Pella (Central Macedonia), Achaia, and Korinthia (Peloponnesus) and in cherry plum in Achaia (Peloponnesus). Six of 11 Japanese plums (cvs. Angeleno, Santa Rosa), 2 of 12 cherry plums, and 8 of 12 peaches (cvs. Spring Crest, Red Haven) examined were found HSVd-infected, but none of the five European plums were. Nucleotide sequence analyses of purified and cloned amplicons from peaches and Japanese and cherry plums revealed sizes of 297 to 308 nt and similarity to sequence variants of other HSVd isolates previously characterized: 95 to 97% identity with the Moroccan isolates apr.9, apr.10, apr.11, and apr.12 and the Spanish isolate apr.4 from apricot (1) (GenBank Accession Nos. AJ297825 to AJ297828 and Y09346, respectively). For confirmation of HSVd presence in field trees, 10 Japanese plums cv. Angeleno, 10 peaches cv. June Gold, and 10 peaches cv. Spring Crest, HSVd-negative (TPH), were bud- or chip-grafted from two of the aforementioned Japanese plums cv. Angeleno and two of the aforementioned peaches cv. Red Haven. Two years later, five Japanese plum trees (cv. Angeleno) and five peach trees (three cv. Spring Crest and two cv. June Gold) were found HSVd-positive with TPH; no fruits were observed to produce fruit symptoms as the grafted trees were kept in an insect-proof greenhouse (no bees for cross-pollination). To our knowledge, our investigation reports for the first time the occurrence of HSVd infecting Japanese plum, cherry plum, and peach in Greece, emphasizing the need for a certification program for the prevention of spreading stone fruit tree viroids in this country. References: (1) K. Amari et al. J. Gen. Virol. 82:953, 2001. (2) N. Astruc et al. Eur. J. Plant Pathol. 102:837, 1996. (3). F. Faggioli et al. Acta. Hort. 550:59, 2001. (4) T. Sano et al. J. Gen. Virol. 70:1311, 1989.

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First Report of the Presence of Tomato apical stunt viroid on Tomato in Sénégal

Plant Disease ◽

10.1094/pdis-91-3-0330c ◽

2007 ◽

Vol 91 (3) ◽

pp. 330-330 ◽

Cited By ~ 6

Author(s):

T. Candresse ◽

A. Marais ◽

F. Ollivier ◽

E. Verdin ◽

D. Blancard

Keyword(s):

Ivory Coast ◽

Polyacrylamide Gel Electrophoresis ◽

Potato Spindle Tuber Viroid ◽

First Report ◽

Stunt Viroid ◽

Reverse Transcription Pcr ◽

New Information ◽

Leaf Chlorosis ◽

Viroid Infection ◽

Leaf Curl Virus

Tomato apical stunt viroid (TASVd) was initially discovered in the Ivory Coast (2). It was later reported in Indonesia and more recently was found to be responsible for severe outbreaks in protected tomatoes in Israel (1) and Tunisia (3). Although not of quarantine status, TASVd is included in the EPPO alert list. In 2005, severe arrest of apical growth and leaf chlorosis were observed in tomato samples from northern Sénégal. Tomato yellow leaf curl virus was initially identified in some samples, but since the symptoms observed were reminiscent of those associated with viroid infection, samples were analyzed by return-polyacrylamide gel electrophoresis and molecular hybridization with a Potato spindle tuber viroid (PSTVd) probe. Positive results prompted a reanalysis by reverse transcription-PCR assays specific for PSTVd or TASVd. Positive amplification was only obtained with the TASVd-specific primers (Vir+ GGGGAAACCTGGAGGAA and Vir- GGGGATCCCTGAAGGAC), and the identity of the viroid confirmed by sequencing of the amplified fragment. The complete genome sequence obtained (GenBank Accession No. EF051631) shows 94 to 96% identity with other TASVd sequences in the databases, the highest homology being with the original Ivory Coast isolate (96%, 11 mutations, and 4 indels for the 362-nt genome). These results provide new information on the diversity of TASVd and of its detrimental potential for tomato crops and represent, to our knowledge, the first report of the presence of TASVd in Sénégal. References: (1) Y. Antignus et al. Phytoparasitica 30:502, 2002. (2) C. R. Walter. Acad. Sci. 292:537, 1981. (3) J. Th. J. Verhoeven et al. Plant Disease 90:528, 2006.

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First Report of Grapevine yellow speckle viroid-1 and Hop stunt viroid Infecting Grapevines (Vitis vinifera) in India

Plant Disease ◽

10.1094/pdis-05-13-0494-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1517-1517 ◽

Cited By ~ 14

Author(s):

A. B. Sahana ◽

C. R. Adkar-Purushothama ◽

G. Chennappa ◽

Z. X. Zhang ◽

M. Y. Sreenivasa ◽

...

Keyword(s):

Sequence Analysis ◽

Vitis Vinifera ◽

Northern Blot ◽

Single Type ◽

Rt Pcr ◽

Thompson Seedless ◽

Stunt Viroid ◽

Blast Analysis ◽

Hop Stunt Viroid ◽

Viroid Infection

During March through July 2012, 10 to 15% of the Vitis vinifera cultivars Thompson Seedless and Anab-e-Shahi exhibited yellow leaf spots and flecks, shortened internodes, and tiny yellow leaves in vineyards of the Bijapur, Doddaballapur, and Kolar districts of Karnataka State, India. These are the major grapevine cultivation regions in India. Samples were collected from four different plants from each district (12 samples in total) and RNA was extracted using 2X CTAB buffer (1). Presence of Grapevine yellow speckle viroid1 (GYSVd-1, genus Apscaviroid) was tested by reverse transcription (RT)-PCR with primer pair PBCVd100C/194H (4) for the amplification of a 220-bp region of the genome. In agarose gel electrophoresis, five samples showed amplicons of the expected size. These amplicons were cloned and sequenced. BLAST analysis confirmed the presence of GYSVd-1. Based on this data, the full-length genome of GYSVd-1 was amplified by RT-PCR using primer pair 341M (5′-CACTCGCGGGGCGCGTTGGA-3′) and 342P (5′-CAATCCCCGGAACCCCCGCT-3′) and the amplicons were cloned and sequenced. Sequence analysis revealed two sequence variants namely Kar-1 (GenBank Accession No. AB742222) and Kar-2 (AB742223) with 98% and 99% identity to GYSVd-1 variants IXc (X87913) and II (X87906), respectively. GYSVd-1 variants Kar-1 and Kar-2 clustered in two distinct phylogenetic sub-clades. All 12 samples also tested positive for Hop stunt viroid (HpSVd, genus Hostuviroid) in two separate sets of RT-PCR using HSV-78P (5′-AACCCGGGGCAACTCTTCTC-3′) and HSV-83M (5′-AACCCGGGGCTCCTTTCTCA-3′); and HSV-7P (5′-AATTCTCGAGTTGCCGC-3′) and HSV-220M (5′-CGAACCGAGAGGTGATGCCA-3′), with the expected size of 303 and 213 bp, respectively (3). Sequence analysis of the amplicons confirmed the presence of HpSVd. Alignment of HpSVd nucleotide sequences obtained from the 12 samples showed the presence of a single type of sequence variant, namely Ind-2 (AB742225). BLAST analysis showed 99% sequence identity of Ind-2 with a HpSVd variant isolated from a 100-year-old grapevine in China. All 12 grapevine samples were also tested for the presence of Australian grapevine viroid (AGVd, genus Apscaviroid), Grapevine yellow speckle viroid 2 (GYSVd 2, genus Apscaviroid), and Citrus exocortis viroid (CEVd, genus Pospiviroid) by RT-PCR as described previously (2). None of the samples showed any positives. Northern blot assay using appropriate digoxigenin-labeled riboprobes performed as described previously (2) further confirmed RT-PCR results. Positive controls for RT-PCR and Northern blot were obtained from viroid-infected grapevines maintained in the greenhouse. GYSVd-1 and HpSVd were detected in symptomatic and symptomless plants. Hence, the symptoms observed in the vineyard cannot be attributed to viroid infection. More work is needed to identify the causal agent(s) of the decline of Thompson Seedless and Anab-e-Shadi cultivars. References: (1) C. R. Adkar-Purushothama et al. Plant Dis. 97:149, 2013. (2) D. Jiang et al. Virus Res, 169:237, 2012. (3) Y. Kawaguchi-Ito et al. PLoS One 4:e8386, 2009. (4) L. I. Ward et al. Plant Dis. 95:617, 2011.

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