Virus and Virus-like Diseases of Pome and Stone Fruits

Chapter 17

Apple chlorotic leaf spot virus in Stone Fruits A. Myrta, S. Matic, T. Malinowski, G. Pasquini, and T. Candresse

Introduction Apple chlorotic leaf spot virus (ACLSV), first isolated from apple trees in the United States after transmission to Malus platycarpa (Mink and Shay, 1959), was subsequently found in other pome and stone fruit trees. The virus name originates from the symptoms induced in the apple clone R 12740-­7A. ACLSV originally was classified in the genus Closterovirus, but is now recognized as the type member of the genus Trichovirus (Martelli et al., 1994).

ACLSV infects all stone fruit crops such as peach, plum, apricot, almond, and cherry. It generally shows a high prevalence (with the notable exception of certified plant material) and has a worldwide distribution (Yoshikawa, 2001). Although latent infection is observed frequently, a variety of symptoms, sometimes severe, have been ascribed to the presence of particular ACLSV isolates or to particular viral isolate–plant cultivar combinations.

ACLSV shows a very large molecular variability and many isolates with different pathogenic characteristics have been described. For instance, some viral isolates from Prunus cannot be transmitted or do not cause symptoms in pome fruit indicators (e.g., R 12740-­7A), whereas some isolates from Malus infect stone fruit trees with difficulty (Chairez and Lister, 1973; Paunovic, 1988).

et al., 1992; Candresse et al., 1996). Replication is presumed to be cytoplasmic and involves the product of ORF1 (Martelli et al., 2000).

High molecular variability exists between ACLSV isolates (Candresse et al., 1995; Al Rwahnih et al., 2004; Yaegashi et al., 2007), especially at the level of nucleotide sequences in the part of the genome where the MP and CP genes overlap. At the protein level, a higher variability is observed in the encoded MP, whereas the CP is more conserved (Candresse et al., 1995; German et al., 1997). The coat protein subunits of different ACLSV isolates show variations in their electrophoretic mobility rates due to variation in amino acid composition rather than to differences in the overall protein size (Malinowski et al., 1998; Pasquini et al., 1998; Al Rwahnih et al., 2004).

The complete nucleotide sequences of ACLSV isolates of plum from France (P863) (M58152, German et al., 1990) and Germany (PBM1) (AJ243438, Jelkmann, 1996), of apple from Japan (P-­205) (D14996, Sato et al., 1993) and (MO-­5 (AB326225), B6 (AB326224) and A4 (AB326223)) (Yaegashi et al., 2007), of cherry from Hungary (Balaton-­1) (X99752, German et al., 1997), and of peach from the United States (EU223295, Marini et al., 2008) are available, together with partial sequences of many isolates and sequence variants, further underlining the large molecular variability of ACLSV.

Taxonomic Position and Nucleotide Sequence

Economic Impact and Disease Symptoms

Family: Betaflexiviridae; genus: Trichovirus; species: Apple chlorotic leaf spot virus (ACLSV) Virus particles are very flexuous filaments 12 × 720 nm in size, with distinct cross banding made up of coat protein subunits of a single type, and encapsidating a single-­stranded, positive-­sense RNA of about 7,500 nt, excluding the poly-­A tail (Yoshikawa and Takahashi, 1988). The virus genome harbors three slightly overlapping open reading frames (ORFs) encoding proteins with approximate molecular weight of 217, 50, and 22 kDa, respectively. ORF 1 codes for a protein that contains signature sequences typical of replicase-­ associated proteins (methyl-­transferase, helicase, and RdRp) of the α-­like supergroup of plant viruses (German et al., 1990). ORF 2 codes for the putative movement protein (MP), and ORF 3 codes for the coat protein (CP) (German et al., 1990). The largest protein (217 kDa) is directly expressed from the genomic RNA, while the other two are expressed via subgenomic mRNAs (German

ACLSV is economically important due to its worldwide occurrence and to the detrimental effects caused in some infected fruit tree species. Some viral isolates are in particular associated with severe fruit deformations and yield reduction (Dunez et al., 1972; Peña-­Iglesias and Ayuso, 1973; Delbos and Dunez, 1988), as well as with graft incompatibility and bud necrosis in nursery production of some cultivars of apricot (Morvan and Castelain, 1967; Marenaud, 1968; Desvignes and Boyé, 1988; Desvignes, 1999).

Symptom severity depends primarily on the plant species and virus isolate (Németh, 1986). Infection is often symptomless in most stone fruit cultivars and with most viral isolates.

When present, symptoms appear mainly on leaves and fruits, and more rarely on the trunk.

In the Mediterranean region, among stone fruit crops, ACLSV was found more frequently in apricot (Myrta et al., 2003). Severe virus isolates elicit a disease of apricot known as

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“pseudopox” (Desvignes and Boyé, 1988), characterized by depressions and protuberances that deform the fruits. Comparable disorders of the same species, called ‘viruela’ and ‘butteratura,’ have also been associated with the presence of ACLSV (Peña-­ Iglesias and Ayuso, 1973; Ragozzino and Pugliano, 1974; Cañizares et al., 2001). In ‘butteratura’-­affected apricot fruits (Fig. 17.1), yellow-­cream spots or depressions (Fig. 17.2) were observed in the stones. In addition to fruit deformation, these diseases are also associated with leaf symptoms, including sometimes severe leaf deformation and chlorotic arabesques generally along the veins that are reminiscent of Plum pox

virus symptoms (Figs. 17.3 and 17.4). When experimentally transmitted to other Prunus such as peach or cherry, the causal isolates induced similar symptoms in these experimental hosts (Desvignes, 1999).

Some ACLSV isolates can induce bark splitting (Dunez et al., 1972, Fig. 17.5) or “false plum pox” symptoms on the fruits and leaves of some plum cultivars (Jelkmann and Kunze, 1995; Lebas et al., 2004, Fig. 17.6). Severe stem pitting or grooving was observed in Japanese plums with mixed infections by ACLSV and Apricot pseudo-­chlorotic leaf spot virus (Liberti et al., 2005), but in this later case the contribution of one or the

Fig. 17.1. ‘Butteratura’ symptoms on apricot cv. Bulida caused by an isolate of ACLSV. Fig. 17.4. Chlorotic leaf symptoms caused by a ‘butteratura’ isolate of ACLSV in apricot cv. Cafona.

Fig. 17.2. Yellow spots and depressions of apricot stones cv.

Bulida affected by a ‘butteratura’ isolate of ACLSV.

Fig. 17.3. Severe leaf deformation and yellow chlorotic symptoms caused by a ‘butteratura’ isolate of ACLSV in peach.

Fig. 17.5. Bark split symptoms caused by the P863 isolate of ACLSV in ‘Prune d’ente’ plum.

Apple chlorotic leaf spot virus in Stone Fruits | 87

Fig. 17.6. Symptoms of ‘false plum pox’ caused by ACLSV in plum.

Fig. 17.8. Symptoms of ‘dark green sunken mottle’ caused by ACLSV in the GF305 peach seedling indicator.

Fig. 17.7. Necrotic symptoms caused on cherries by a severe isolate of ACLSV.

other of these two agents to the symptoms observed remains to be clarified.

Sharka-­like symptoms on the fruits (plum pseudopox) and chlorotic line pattern on the leaves of ACLSV-­infected peaches (Fig. 17.3), and necrosis of the fruits, bark splitting and decline in some sweet cherry varieties have also been observed (Desvignes and Boyé, 1988; Németh, 1986, Fig. 17.7). However, in peach, the most frequent symptoms are dark green punctuations or depressions on the surface of the leaves, for which the name “dark green sunken mottle” (Delbos and Dunez, 1988; Desvignes, 1999, Fig. 17.8) has been coined. This is the reaction observed in the GF305 peach seedling indicator upon inoculation with the majority of ACLSV isolates.

In addition, ACLSV, together with Prunus necrotic ringspot virus, may give rise to chlorotic “oak leaf like” patterns, observed on plum or peach leaves. Even in situations where it is latent, ACLSV infection is considered by some authors to act synergistically to enhance the severity increase of the symptoms caused by other pathogen infection or even by environmental abiotic stress such as mineral deficiencies (Desvignes, 1999).

Host Range ACLSV infection has been recorded from a wide range of cultivated and ornamental Prunus species (Giunchedi, 2003; Myrta et al., 2003). All important cultivated Prunus crops appear susceptible, including peach, apricot, almond, sour and sweet cherry, and domestic and Japanese plum. Other Prunus species for which natural infection has been reported include blackthorn (P. spinosa, Sweet, 1980), Himalayan wild cherry (P. cerasoides, Rana et al., 2008) and dwarf flowering almond (P. glandulosa ‘Sinensis,’ Spiegel et al., 2005).

ACLSV is also able to infect a range of pome fruit species, including apple, pear, and quince (see chapter 4 in this book) in which it frequently shows significant prevalence. It also infects a number of wild or ornamental apple species and has also been detected in natural infection in other rosaceous species such as hawthorn (Sweet, 1980) and medlar.

ACLSV has a very limited experimental herbaceous host range, which includes several useful experimental hosts such as Chenopodium quinoa, C. amaranticolor, and Nicotiana occidentalis (Lister et al., 1965; Yoshikawa, 2001).

Transmission ACLSV is readily transmitted by grafting of woody host material or by vegetative propagation (Németh, 1986). It can be transmitted with some difficulty by mechanical inoculation from woody hosts to herbaceous hosts and, more readily, between herbaceous hosts.

No natural vectors are currently known (Yoshikawa, 2001) and, similarly, ACLSV is not known to be seed-­or pollen-­ transmitted in any of its hosts (Yoshikawa, 2001).

The presence of multiple isolates of the virus in the same plant has been taken as suggesting the existence of an unidentified natural mode of transmission (Pasquini et al., 1998) but other explanations are also possible, such as the impact of cultural practices (top grafting, rootstock and culitvar infections, etc.). Rare cases of apparent field spread have sometimes been ascribed to natural root grafts between trees.

Geographical Distribution and Epidemiology ACLSV has a worldwide distribution (Delbos and Dunez, 1988, Yoshikawa, 2001) and is probably present wherever susceptible fruit tree species are grown. Since the virus does not appear to spread naturally, the presence of mostly latent infections in the majority of fruit tree cultivars and the use of infected propagation material are regarded as the main ways for its dissemination.

88 | Chapter 17

Detection ACLSV can be detected by indexing onto sensitive woody indicators under greenhouse conditions, i.e., peach GF305 grown at 20–25°C for 8 weeks, and Prunus tomentosa IR473 × IR474 hybrid grown at 22°C for 12 weeks (Jelkmann, 2001, 2004).

Mechanical transmission is possible; indexing on herbaceous indicators is also possible, with the recommended indicator being Chenopodium quinoa grown at 20°C for 20 days. Both local and systemic symptoms appear on C. quinoa infected with ACLSV (Jelkmann, 2004). However, indexing on C. quinoa is clearly a technique of lower sensitivity and the samples to be indexed should be carefully selected since both the virus titer and the presence of inhibitors are known to vary depending on the type of tissue sampled and on the time of the year. In particular, since the virus titer is generally lower during the hot summer season, spring samples should be preferred (Candresse et al., 1995; Kummert et al., 2004). Petals and young leaves are the preferred source tissues.

The virus is moderately immunogenic. It can be detected serologically, either by ELISA (Flegg and Clark, 1979), immuno tissue printing (Knapp et al., 1995), or immuno electron microscopy (Kerlan et al., 1981; Kalashjan and Lipartia, 1986).

Because of the nature of the material to be assayed, the heterogeneity of the repartition in the plant of the viral isolates, and of the low virus titer in the tissues, difficulties can be met with standard ELISA testing. To overcome detection problems, a modified ELISA procedure in which the enzyme conjugate was incubated simultaneously with plant extracts increased the sensitivity of detection (Flegg and Clark, 1979). Also, F(abʹ) 2 ELISA is quite effective for ACLSV detection (Barba and Clark, 1986). There are considerable differences in virus titer in different plant tissues. The highest antigen concentration was found in cortical scrapings of one-­to two-­year-­old cuttings, flower petals (from naturally developed buds, non-­forced shoots), and mature fruits (Barba and Clark, 1986; Cieślińska et al., 1995; Llácer et al., 1985). In areas with temperate climate, the most favorable period for ELISA detection is from early May to late June (Karešová and Paprstein, 2001). Cieślińska et al. (1995) reported that testing cherry and plum was effective in June and July, but not earlier in April and May. Different polyclonal reagents for virus detection are commercially available, whereas for more specific detection monoclonal antibodies are used (Poul and Dunez, 1989, 1990; Malinowski et al., 1997).

The availability of ACLSV sequences has allowed the development of molecular detection tools such as RT-­PCR. To overcome the inhibition of reaction often caused by phenolic compounds or polysaccharides, an immunocapture (IC)-­RT-­ PCR was developed, with the sensitivity of detection down to 10 fg of purified virus (Candresse et al., 1995; Nemchinov et al., 1995). A polyvalent nested RT-­PCR using degenerate and inosine-­containing primers has been developed for detection of Tricho-­, Capillo-­, and Foveaviruses, including ACLSV (Foissac et al., 2001). Although these techniques are very sensitive, they may not detect all ACLSV isolates due to the high molecular variability of the virus (Al Rwahnih et al., 2004; Malinowski, 2005; Yaegashi et al., 2007). Menzel et al. (2002) designed a new set of primers for amplifying a fragment of the ACLSV genome, used in conjunction with amplification of plant mRNA as internal control. Salmon et al. (2002) and Roussel et al., (2005) developed real-­time 5ʹ nuclease RT-­PCR detection using a fluorogenic 3ʹ minor groove binder-­DNA probe, which is particularly suitable for the detection of agents with high molecular variability between strains.

Multiplex RT-­PCR has been used for the simultaneous detection of two (Menzel et al., 2002) and four viruses (Menzel et al., 2003; Hassan et al., 2006), including ACLSV and simulta-

neous detection of six stone fruit viruses, including ACLSV, by molecular hybridization using a polyprobe (Herranz et al., 2005). A recent comparison has allowed the comparisons of various PCR-­based assays and of other detection techniques, in terms both of polyvalence and of sensitivity, demonstrating the interest of PCR-­based assays (Spiegel et al. 2006).

Control The most effective way of control is the use of virus-­free propagating material. Thermotherapy or shoot-­tip grafting have been successfully used for elimination of ACLSV (Navarro et al., 1982). Chemotherapy, alone or in combination with thermotherapy, has also been used with success for the elimination of ACLSV from cherry (Deogratias et al., 1989) as well as from pome fruit materials (Hansen and Lane, 1985). Sanitary selection and sanitation in the framework of a certification program are liable to achieve durable results.

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Chapter 18

Apple mosaic virus in Stone Fruits S. Paunovic, G. Pasquini, and M. Barba

Introduction

(Murphy et al., 1995). It is a quasi-­isometric, multicomponent, divided genome virus, with at least three nucleoprotein particle types. ApMV has the same genome organization as other Ilarviruses, encoding functionally similar translation products, as those of other Bromoviridae members such as Bromovirus, Cucumovirus, and Alfamovirus (with the Alfalfa mosaic virus -­ AMV, as the sole member of the genus). It has three positive-­ sense single stranded genomic RNAs, designated RNA 1, 2, and 3, and an RNA 4 which is a subgenomic messenger for the coat protein (CP) of the virus. RNAs 1 and 2 each contain one single open reading frame (ORF) and encode non-­structural proteins (P1 and P2) involved in viral RNA synthesis (Shiel and Berger, 2000). Unlike all Ilarviruses that have been sequenced, ApMV does not contain a second ORF 2b on RNA2, and is similar to AMV in that respect. According to sequence similarities of RNAs 1 and 2, ApMV appears to be more closely related to AMV and Prune dwarf virus (PDV) than to other viruses of the Bromoviridae family. The mixture of the three genomic RNAs is not infectious, and the presence of either subgenomic RNA 4 or of the CP (homologous ApMV CP or a heterologous CP from other Ilarviruses [but not from other genera]), is required for genome activation and for the initiation of infection. RNA 3 has two ORFs, which code the movement protein (P3a) and the CP. The P3a protein is translated directly from genomic RNA 3, whereas the CP is translated from subgenomic RNA 4 (Guo et al., 1995).

The complete nucleotide sequences of the CP gene were determined for several ApMV isolates originated not only from Malus spp., Pyrus spp., and hop, but also from Prunus spp. (almond and plum). The CP gene of the ApMV-­G isolate, originating from Prunus mahaleb L., is 657 nt in length and encodes protein of 218 aa with a predicted molecular mass of 24.6 kDa. The CP gene of this isolate shares 87.3% identity at the nucleic acid level with that of isolate ApMV-­I (originating from apple) with three one-­base insertions in the ApMV-­G CP gene. These insertions lead to a frameshift of 12 amino acids in comparison with ApMV-­I (Guo et al., 1995).

ApMV isolates originating from Prunus species can show significant size variability at the last 400 nt of their RNAs 3 and 4 (Saade et al., 2000). For example, the nucleotide sequence analysis of two ApMV isolates revealed the existence of a 28-­nucleotide deletion in the 3ʹ-­untranslated region (3ʹ-­UTR) in comparison with reference isolate from apple.

Comparison of the nucleotide sequences of the CP gene of 11 European and American isolates also showed remarkable variability in the 5ʹ-­half of the gene: 94 positions out of 336 are variable, compared with 61 positions out of 336 in the 3ʹ-­half (Petrzik and Lenz, 2002). Two American apple isolates (A and

Apple mosaic virus (ApMV) is the causal agent of several diseases of the line pattern type affecting most cultivated Prunus spp., including plum, almond, peach, apricot, cherry and sour cherry. However, similar symptoms on Prunus spp., Japanese plum, peach and flowering cherry in particular, may also be caused by other Ilarviruses, e.g., Prunus necrotic ringspot virus (PNRSV) and American plum line pattern virus (APLPV) (see pertinent chapters in this volume).

Atanasoff (1935) was the first to describe mosaic diseases on stone fruits in Bulgaria, and by 1970 the occurrence of similar diseases in Prunus spp. had been reported under the same or different names, i.e., striped variegation, oak-­leaf pattern, banded chlorosis, yellow band mosaic or line pattern in many, mostly Euro­pean, countries (Arnaud and Arnaud, 1936; Josifovic, 1935; Christoff, 1938; Willison,1945; Kirkpatrick, 1955; Gilmer, 1956; Posnette and Ellenberger, 1957; Ellenberger, 1962; Nemeth, 1986).

The possible relationships between virus-­caused line pattern diseases and ApMV were indicated by the reports of interspecies inoculations of those viruses: isolates from plum, peach and cherry with line pattern symptoms produced mosaic in apple, and inoculums from apple produced line pattern in plum (Christoff, 1938; Kirkpatrick, 1955; Gilmer, 1956; Posnette and Ellenberger, 1957; Fulton, 1965). The transmission of the virus from plum to herbaceous plants, its back-­inoculation on Prunus spp. and the subsequent virus identification by purification and study of its electron-­microscopic and serological properties firmly suggested close relationship between the virus causing line patterns and ApMV (Seneviratne and Posnette, 1970).

Following the proposal of Seneviratne and Posnette (1970), the disease was also called European plum line pattern for the purpose of avoiding possible confusion with American plum line pattern which is caused by another Ilarvirus (Paulsen and Fulton, 1968; Fulton, 1982; Scott and Zimmerman, 2001), not serologically related to ApMV. ApMV infections of different Prunus spp. grown worldwide are confirmed by serological and modern molecular methods.

The virus is more common on Prunus spp. than on Malus spp. in Europe, where it seems to be spread by vegetatively propagated tolerant plum rootstocks like damson, greengage, Pollizo, etc. (Desvignes et al., 1999).

Taxonomic Position and Nucleotide Sequence Family: Bromoviridae; genus: Ilarvirus; species: Apple mosaic virus. Apple mosaic virus is a member of the subgroup III of the genus Ilarvirus, which belongs to the family Bromoviridae

91

92 | Chapter 18

I) and two European almond (Venza) and apple (H) isolates were identified to have insertions of 6 nt to 15 nt after nucleotide position 141, in comparison with consensus apple isolates sequence. The almond isolate has a CP 220 aa long, with 2 aa additions due to the insertions of 6 nt (9 nt insertion and deletion of a consensus AAG codon). The length of the CP gene of an isolate from plum sequenced in the same study was 218 aa and was the same as that for the previously published isolate from P. mahaleb.

Economic Impact The presence of ApMV in stone fruits is recorded occasionally. The infection may result in growth reduction and yield losses in susceptible cultivars (Nemeth, 1986; Diekmann and Putter, 1996), however, no precise information on economic losses is available. ApMV infection also exerts a detrimental effect on leaf content of bittering acids in hops, decreasing alpha acid yields by 5–34%, can cause important yield reduction in hazel (Aramburu and Rovira, 1995; Aramburu and Rovira, 2000), and causes reduction of apple bud take in nurseries (Nemeth, 1986; Crowle et al., 2003).

In some Mediterranean countries, ApMV is economically important on almond. In particular, old cultivars are heavily affected by the virus in mixed infections with other viruses and diseased plants decline and show yield reduction.

Symptoms The leaves of infected trees almond, plum, apricot, peach, and cherry show typical yellow line pattern, bright yellow blotches, rings, bright yellow vein clearing, and oak-­leaf pattern (Figs.

18.1 and 18.2) (Posnette and Ellenberger, 1957; Canova, 1960; Ellenberger, 1962; Nemeth, 1986; Diekmann and Putter, 1996).

Symptoms generally appear at the beginning of summer and, in some cases, are present only on a limited number of leaves randomly distributed on the plants.

Bright chrome yellow discolorations on leaves in the form of patchy or more or less widespread mottling, ringspot and line patterns are caused by the presence of ApMV in almond (Savino et al., 1995) (see pertinent chapter in this volume). In some sensitive almond cultivars, the virus induces failure of blossom and leaf buds to grow, a symptom known as almond leaf failure (Diekmann and Putter, 1996; Desvignes et al., 1999).

The symptomatology is generally not of diagnostic significance, because similar symptoms may be produced on Prunus spp. by other Ilarviruses, e.g., PNRSV and APLPV. Moreover, some peach cultivars may fail to display any symptoms (Choueiri et al., 2001), so that laboratory tests are needed for the reliable identification of the virus.

Host Range The natural host plants of ApMV are various Prunus spp., such as P. armeniaca L., P. cerasifera L., P. domestica L., P. instititia L., P. mahaleb L., P. persica L., P. salicina L., P. serrulata L., P. amygdalus L., P. triloba L., P. cerasus L., and P. avium L. ApMV also occurs naturally in Malus spp., Rubus spp, Rosa spp., birch (Betula spp), hop (Humulus lupulus), horse chestnut (Aesculus hippocastanum) and red horse chestnut (A. carnea), hazelnut (Corylus avellana) and Chenomeles japonica (Sweet, 1980; Nemeth, 1986; Diekmann and Putter, 1996; Desvignes et al., 1999; ICTVdB, 2002). Experimentally, over 65 herbaceous plant species in 19 families are susceptible (ICTVdB, 2002). Among these, several diagnostic hosts are used as indicators in biological tests (EPPO, 2000).

Transmission No insect vector is known for ApMV and the virus is only transmitted by vegetative propagation of rootstocks and planting material from infected mother trees, and by graft-­inoculation of woody plants. The virus can be sap-­transmitted by mechanical inoculation, but not easily, to several herbaceous plants such as Cucumis sativus, Petunia hybrida, Chenopodium quinoa, C. amaranticolor, Cucurbita maxima, C. pepo, Nicotiana benthamiana, and N. megalosiphon. Petal extracts prepared in phosphate buffer containing antioxidants such as sodium diethyldithiocarbamate, sodium thioglycolate, and ascorbic acid represent the best inoculum for mechanical transmission.

ApMV has not been detected in pollen grains from infected plum and apricot trees, either externally or internally (Digiaro et al., 1992), which suggests that it is not pollen-­transmissible.

Similarly, the results of Barba et al. (1986) proved that ApMV is not transmissible by seeds of infected almond trees, in agreement with the findings of Sweet (1980) who suggested that ApMV is not seed-­transmissible in Aesculus species.

Geographical Distribution and Epidemiology ApMV is distributed worldwide, more commonly on Prunus spp. than on Malus spp. in Europe. It is often found in mixed infection with PNRSV and PDV, but the frequency of ApMV infection seems to be much lower than of these two other viruses, especially in plum, apricot, peach, and cherry (Petrzik and Lenz, 2002; Myrta et al., 2003).

Fig. 18.1. Diffuse yellow discolorations on leaves of an almond plant naturally infected with ApMV.

Fig. 18.2. Yellow line pattern on cherry leaves infected with ApMV.

Apple mosaic virus in Stone Fruits | 93

The average infection rates of Mediterranean stone fruit by ApMV recorded after a survey conducted during the 1990s were 4.0%, 3.0%, 3.2%, 3.3%, and 1.1% in apricot, peach, almond, cherry, and plum, respectively (Myrta et al., 2003).

However, the presence of ApMV in almond was reported to be as high as 45% in southeastern Italy (Savino et al., 1995; Di Terlizzi, 1998) and 14–17% in the Valencia and Murcia regions of Spain (Llacer et al., 1986; Pallas et al., 1998). In addition, the results of the survey conducted for the virus prevalence on plum, apricot, and peach in the above-­mentioned regions were 21%, 20%, and 2%, respectively, in Italy (Di Terlizzi et al., 1992), 7% for both apricot and plum, and 1% for peach in Valencia (Llacer et al., 1986). Also, in the Murcia region, the level of ApMV infection of apricot was reported to be relatively high, i.e., 15.7% (Dominguez et al., 1998).

Detection ApMV can be detected by grafting onto woody indicator plants such as GF305 peach seedling or peach cv. ‘Elberta’ in the field, but testing in a greenhouse with controlled air conditions is recommended (Desvignes, 1976, EPPO, 2000). Three months are required for the development of symptoms under greenhouse conditions (20°C), whereas in the field, it can take about 2 years.

Both inoculated indicator plants display light green, yellowish, or bright yellow rings, spots, bands, or oak-­leaf patterns on leaves (Fig. 18.3) (Desvignes, 1976; Nemeth, 1986).

The herbaceous indicator plants used for biological indexing following mechanical inoculation are C. quinoa, C. amaranticolor, C. sativus, P. hybrida, and C. pepo. Sap-­inoculation on P. hybrida results in systemic lines and gray concentric rings, whereas C. quinoa reacts with irregular necrotic lesions both on the inoculated and later developed leaves. C. sativus, the most susceptible herbaceous host plant, reacts to the infection with large chlorotic primary lesions on the cotyledons and systemic yellow vein banding and stunting (Fig. 18.4). Biological indexing on herbaceous indicators is, however, of limited sensitivity and therefore not reliable for virus detection, and laboratory tests are additionally required for the final identification of the virus.

Originally, the virus was detected serologically by gel-­ diffusion test (De Sequeira, 1967), but more recently, reliable, sensitive and rapid testing by DAS-­ELISA is applied, allowing high throughput analysis (Clark and Adams, 1977; Barbara et al., 1978; Torrance and Dolby, 1984). The highest virus concentration is usually found in forced buds (leaf and flower buds), in

Fig. 18.3. Line pattern symptoms on a GF305 peach seedling experimentally chip budded with ApMV infected tissue.

naturally grown petals, as well as in young leaves which are the best samples for routine testing. However, the bark of one-­year old shoots can also be used. The virus could be detected less readily in mature plum leaves after June than either PNRSV or PDV (Torrance and Dolby, 1984).

Monoclonal antibodies have been produced from an ApMV almond isolate from Italy and used to develop an indirect ELISA assay for the virus routine diagnosis from stone fruit samples (Pasquini and Barba, 1991). The specific monoclonal antibodies produced against an ApMV isolate from P. domestica ‘Ada Bayer’ have been used in indirect ELISA concurrently with monoclonal antibodies against other Ilarviruses to serotype a panel of isolates (Halk et al., 1984).

Determination of the sequence of the CP gene of ApMV (Alrefai et al., 1994; Sanchez-­ Navarro and Pallas, 1994; Candresse et al., 1998) has provided the base for sensitive molecular detection of the virus by reverse-­transcription polymerase chain reaction (RT-­ PCR). ApMV can be detected after immobilization of plant extracts on a solid support by direct binding-­PCR (DB-­PCR) system (Rowhani et al., 1995).

Candresse et al. (1998) developed immuno-­ capture PCR-­ ELISA procedure for the simultaneous detection and identification of ApMV and PNRSV. Tissue print hybridization has successfully been used to detect the virus from winter bark (Matic et al., 2008) and a non-­isotopic molecular hybridization assay developed to detect simultaneously the four Ilarviruses affecting stone fruits (ApMV, PNRSV, PDV, and APLPV), together with Apple chlorotic leafspot virus (ACLSV) and Plum pox virus (PPV).

The protocols developed used either a ‘polyprobe four’ for Ilarviruses and a ‘polyprobe six’ for all viruses (Herranz et al., 2005). Non-­isotopic molecular hybridization and multiplex RT-­ PCR have been compared to provide simultaneous detection of three Ilarviruses that affect Prunus: ApMV, PNRSV, and PDV (Saade et al., 2000). The latter technique was advantageous, combining antisense degenerate primers designed to the 3ʹ-­untranslated region of RNAs 3 and 4 of these viruses and virus-­specific sense primers. The technique allowed not only the simultaneous detection but also identification of the three viruses in a single assay. This PCR-­based technology is several times more sensitive than ELISA, and at least 125 times more sensitive than multiplex nucleic acid hybridization.

Fig. 18.4. Systemic yellow vein banding on Cucumis sativus sap-­inoculated with ApMV infected leaves.

94 | Chapter 18

Sanchez-­Navarro et al. (2005) have developed a sensitive and reliable one-­step RT-­PCR reaction with internal control for the detection and differentiation of eight stone fruit viruses, i.e., ApMV, PNRSV, PDV, APLPV, PPV, ACLSV, Apricot latent virus (ApLV), and Plum bark necrosis and stem pitting associated virus (PBNSPaV). ApMV has been detected using this multiplex RT-­PCR assay in infected field samples of peach, almond, apricot, and GF 305.

The recent technology of oligonucleotide microarray hybridization has been also applied for the detection of ApMV, together with other fruit viruses (ASPV, PDV, PNRSV, and PPV).

The sensitivity of the microarray detection was compared for different kinds of fluorescently labeled targets and the indirect labeling method showed the highest specificity (Lenz et al., 2008)

Control The removal of infected trees and the utilization of certified planting material (EPPO, 2000) are effective control measures, because no natural vectors are known and pollen and seed transmission have not been demonstrated. Virus-­free plants can be produced by thermotherapy of infected plants at 37°C for 3–4 weeks as well as by in vitro shoot tip grafting (Navarro et al., 1988; Barba et al., 1992).

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Fulton, R. W. 1982. Ilar-­like characteristics of American plum line pattern virus and its serological detection in Prunus. Phytopathology 72:1345-­1348.

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Guo, D., Maiss, E., Adam, G., and Casper, R. 1995. Prunus necrotic ringspot ilarvirus: nucleotide sequence of RNA3 and the relationship to other ilarvirus based on coat protein comparison. J. Gen. Virol. 76:1073-­1079.

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Apple mosaic virus in Stone Fruits | 95 Myrta, A., Di Terlizzi, B., Savino, V., and Martelli, G. P. 2003. Virus diseases affecting the Mediterranean stone fruit industry: a decade of surveys. Pages 15-­23 in: Virus and Virus-­like Diseases of Stone Fruits, with Particular Reference to the Mediterranean Regions.

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Pallas, V., Llacer, G., and Cambra, M. 1998. Sanitary status of stone fruit industry in the Mediterranean countries: Spain. Pages 61-­ 64 in: Stone Fruit Viruses and Certification in the Mediterranean Countries: Problems and Prospects. A Myrta, B. Di Terlizzi, and V. Savino, eds. Options Mediterr. Ser. B, 19, CIHEAM. Pasquini, G., and Barba, M. 1991. Production and application of monoclonal antibodies against Apple mosaic virus. Petria 1:31-­36.

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Chapter 19

Apricot latent virus L. G. Nemchinov, P. Gentit, E. Zemcic, T. Candresse, and A. Hadidi

Introduction

al., 2005; Usta et al., 2007). Thus, the previously very limited research interest to the virus and the limited informations on ApLV’s biological properties, geographic distribution, and economic importance are expanding fast, which will undoubtedly facilitate the development of efficient means of its control.

Apricot latent virus (ApLV) was first described in Moldova, former USSR, from latently infected apricot cv. ‘Silistra 4’ introduced from Bulgaria (Zemcic and Verderevskaya, 1993).

During the summer of 1990 peach seedlings graft-­inoculated with apricot chip-­buds from cv. ‘Silistra 4’ developed multiple chlorotic dots 6 months after grafting, which eventually merged into bigger spots. The symptoms were similar to those of Peach asteroid spot and Rusty mottle viruses (Zemcic and Verderevskaya, 1993). Threadlike virus particles, observed in leaf extracts of the inoculated peach seedlings, reacted positively with an antiserum against Apple stem pitting virus (ASPV) (Zemcic et al., 1998). Zemcic and Verderevskaya (1993) suggested possible relation of the discovered virus with ASPV. They nevertheless excluded the assignment of the new virus, tentatively named Apricot latent virus (ApLV), as a strain of ASPV since cross infection was not observed in experiments where peach or apple trees were inoculated with either ASPV or ApLV (Zemcic et al., 1998).

The new virus properties and taxonomic position remained uncharacterized until the 3ʹ terminal sequence of its genome was determined and sequence comparisons allowed ApLV to be assigned as a new, independent viral species in the genus Foveavirus (Nemchinov et al., 2000).

In France, two graft-­transmissible diseases of previously unknown etiology, peach asteroid spot disease (PAS) and peach sooty ringspot disease (PSRS) were found to be caused by agents closely related to ApLV (Gentit et al., 2001a, 2001b).

Peach asteroid spot disease was originally described in the 1930s in the United States, causing small, star-­shaped spots on peach leaves (Cohrnan and Smith, 1938). During a large scale survey conducted in 1982 in apricot orchards in south France, a graft-­transmissible pathogen inducing symptoms on peach similar to those described for peach asteroid spot disease was found on cv. ‘Bebeco.’ Another disease of unknown etiology was reported in apricot cultivars from France and Italy (Grasseau et al., 1999). Since the symptoms resembled those described for quince sooty ringspot, the disease was named peach sooty ringspot (Grasseau et al., 1999). Agents related to ApLV were identified in Prunus materials infected with these diseases and partial genomic sequencing indicated that these agents should be regarded as isolates or strains of ApLV (Gentit et al., 2001b). The direct demonstration that the PAS and PSRS strains of ApLV are indeed responsible for the PAS and PSRS diseases, however, remains to be established.

Lately, the widespread distribution of ApLV was additionally confirmed by its discovery in the Palestine Authority and Turkey (Abadi et al., 2003; Abou Ghanem-­Sabanadzovic et

Taxonomic Position and Nucleotide Sequence ApLV and its two variants observed in peach (PASV and PSRSV) are assigned to the recently established genus Foveavirus with ASPV as type species (Martelli and Jelkmann, 1998). ApLV and ASPV show a similar genomic organization as well as substantial sequence homologies. Indeed, the amino acid (aa) identity between CP of ASPV and that of all known variants of ApLV is as high as ~53–60%, which may explain reports on serological cross reactions between ApLV-­infected Prunus materials and ASPV antisera (Nemchinov and Hadidi, 1998).

By contrast, much lower identity levels, in the 25–35% range, are observed between the CP of ASPV and those of other Fovea or Fovea-­like viruses such as Rupestris stem pitting associated virus (RSPaV, Meng et al., 1998), Cherry green ring mottle virus (CGRMV, Zhang et al., 1998), or Cherry necrotic rusty mottle virus (CNRMV, Rott and Jelkmann, 2001). Similarly, the amino acid identity levels between the triple gene block (TGB) proteins of ApLV (based on the sequence of the PSRSV variant) and the comparable proteins of ASPV are very high, reaching 91%, 77%, and 74% for the TGB1, TGB2, and TGB3 proteins, respectively (Gentit et al., 2001b). By comparison, much lower values are again observed for ASPV, CGRMV or CNRMV. At the moment, four partial genomic sequences of ApLV variants are available in the Genebank international databank: • ApLV original isolate from Moldova (AF057035, 1,444 nt long 3ʹ terminal sequence, including the complete CP gene).

• Peach asteroid spot (PAS) isolate LA2 of ApLV from France (AF318061, 1,112 nt long 3ʹ terminal sequence, including the complete CP gene).

• Peach sooty ringspot (PSRSV) isolate Caserta12 of ApLV from Italy (AF318062, 3,065 nt long 3ʹ terminal sequence, including the complete TGB1-­TGB2-­TGB3 and CP genes).

• ApLV isolate Apr47 from Palestine Authority (AY697862, 200 nt sequence fragment of the CP gene).

The PASV and Palestine Authority isolates show a high degree of sequence identity with the original ApLV isolate from Moldova, which made it possible to classify them as members of the ApLV species. The PSRSV isolate is clearly more divergent and for some proteins, identity levels close to the species

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discrimination border for the genus Foveavirus are observed.

It should nevertheless probably be considered as a distant strain of ApLV rather than as a distinct virus (Gentit et al., 2001a, b).

Economic Impact Due to the generally mild or asymptomatic infection course and the lack of precise information on the full extent of its geographic distribution, ApLV is currently considered as a pathogen of minor economic importance. There are, however, reports that some strains causing the PAS syndrome may kill the affected trees (Nemeth, 1986).

Symptoms ApLV is mostly latent in the natural host from which it was named, apricot. Interestingly though, withered branches were occasionally noticed in ApLV-­ infected trees of the Silistra apricot cultivar 5 years after planting (E. Zemcic, unpublished observation). On apricot cv. Bergeron A660, 2–3 years following the inoculation, the virus induced chlorotic mottles and ringspots along the veins of leaves of a few scattered branches (Fig. 19.1). The Caserta 12 PSRS isolate induced a slight wilting of branches and chlorotic, rolled leaves upon artificial inoculation on apricot cv. Orangered (Fig. 19.2).

On inoculated GF305 peach seedlings, ApLV causes bright, yellowish-­green spots or sometimes large chlorotic blotches randomly scattered on the leaf surface (Fig. 19.3). On peach cultivars Springtime and Elberta inoculated by bud grafting, the original ApLV isolate as well as the PASV LA2 isolate induce chlorotic sooty ringspots on green leaves that remain as green areas as the leaves start turning yellow and senescing (Zemcic and Verderevskaya, 1993; Nemchinov et al., 2000; Abou Ghanem-­Sabanadzovic et al., 2005) (Fig. 19.4). These symptoms resemble to those described for the peach asteroid spot disease in the United States (Williams et al., 1976). In the same host, the PSRSV Caserta 12 isolate causes chlorotic or yellowish and rather diffuse spots scattered around the secondary and tertiary veins. Two weeks later, veins and chlorotic mottles become encircled by a blackish necrotic spotting (Grasseau et al., 1999) (Fig. 19.5). At the ripening stage, peach fruits of cv. ‘Springtime’ inoculated by the original ApLV from Moldova displayed discolored asteroid spots (P. Gentit, unpublished observation) (Fig. 19.6).

Fig. 19.1. Symptoms induced by the original ApLV isolate from Moldova two to three years following inoculation of Prunus armeniaca cv. Bergeron A660. Leaves displayed chlorotic mottles and ringspots along the veins. A leaf from a healthy control plant is shown on the upper left side.

Sweet cherry (P. avium) cultivars ‘Bing,’ ‘Canindex,’ and ‘Sam’ have been reported to develop symptoms in the form of red to purple rings and mottling after chip-­budding of infected peach material (Abou Ghanem-­ Sabanadzovic et al., 2005). Occasional chlorotic-­ green spots, slightly similar to those observed on ApLV-­infected GF305 peach seedlings, appear on the leaves of inoculated P. cerasifera (myrobalan plum) (Nemchinov et al., 2000).

Upon inoculation, the herbaceous indicators, Nicotiana occidentalis ssp. oblique N1 and Nicotiana occidentalis 37B, develop local necrotic spots and vein discoloration, respectively, when mechanically inoculated with ApLV-­infected peach extracts. PASV-­inoculated N. occidentalis plants show mild vein clearing, whereas N. occidentalis inoculated with PSRS exhibit pronounced vein clearing with necrotic spots and apical necrosis (Fig. 19.7) (Gentit et al., 2001a).

Fig. 19.2. Slight wilting of branches and chlorotic, rolled leaves observed upon inoculation of Prunus armeniaca cv Orangered with the peach sooty ringspot (PSRS) isolate Caserta 12.

Fig. 19.3. Symptoms caused by the original ApLV isolate from Moldova on leaves of GF305 peach seedlings. Symptoms are either small bright yellow blotches (center) or larger chlorotic blotches randomly scattered on the leaf surface (right). The leaf shown on the left is from a healthy, uninoculated control plant.

Apricot latent virus | 99

Host Range and Transmission Woody host range. The original ApLV isolate from Moldova was transmitted successfully by grafting to Prunus persica GF 305, P. cerasifera, and P. armeniaca cv. Bergeron A660 (see above). The Palestinian Authority isolate of ApLV was transmitted by chip-­budding to several peach cultivars, to cherry cultivars ‘Bing’ and ‘Canindex,’ and to apricot cultivars Luizet, Priana, and Tilton, which remained symptomless

Fig. 19.6. Discolored asteroid spots caused by the original ApLV isolate from Moldova on fruits of peach cv. Springtime.

Fig. 19.4. Symptoms induced by the original ApLV isolate from Moldova on peach cv. Springtime upon orchard inoculation. Symptoms on yellowing leaves that are beginning to senesce appear as green sooty ringspots.

Fig. 19.7. Local necrotic spots and vein discoloration caused on Nicotiana occidentalis 37B by ApLV isolates. 1a, 1b: PAS isolate LA2. 2a, 2b: Original ApLV isolate from Moldova. 3: PSRS isolate Caserta 12.

Fig. 19.5. Symptoms of peach sooty ringspot (PSRS) isolate Caserta 12 on Prunus persicae cv. Springtime in orchard. Symptoms are chlorotic mottles encircled by a blackish necrotic spotting appearing during the senescence.

Fig. 19.8. RT-­PCR detection of ApLV using the three pairs of virus-­specific primers described in the text. A: H-­ALV1/C-­ALV-­ 1(expected product size 200bp). M, Bio Low Molecular weight marker (Bio Ventures Inc., TN): 1000, 700, 525, 500, 400, 300, 200, 100, and 50 bp, respectively; 1, PCR product amplified from ApLV-­ infected peach seedling GF305; 2, uninfected control. B: H-­ALV2/ C-­ALV2 (expected product size 242 bp) M, Bio Low Molecular weight marker; 1, water control; 2, uninfected GF-­305; 3, chipbud inoculated peach GF-­305; 4, original ApLV-­infected peach from Moldova. C: H-­ALV3/C-­ALV3 (product size 491 bp). M, BioLow molecular weight marker; 1, uninfected tissue; 2, ApLV-­infected peach tissue.

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(Abou Ghanem-­Sabanadzovic et al., 2005). Natural host plants reported for the American peach asteroid spot disease isolates include P. persica, P. andersonii, P. avium, P. armeniaca, P. amygdalys, P. domestica, P. myrobalana, P. salicina, P. spinosa, P. mume, P. mahaleb, P. bokhariensis, and P. angustifolia (Wagnon et al., 1963; Nemeth, 1986). PAS and PSRS isolates found in France and Italy are experimentally transmissible to peach, apricot, and sweet cherry (Gentit et al., 2001a).

Herbaceous host range. As indicated above, the original ApLV isolate from Moldova could be mechanically transmitted using sap from infected peach leaves to N. occidentalis B37 and N. occidentalis ssp. oblique N1 (Zemcic et al., 1998).

Transmission to N. occidentalis B37 was also reported for PASV and PSRSV (Gentit et al., 2001a) and for the Apr47 Palestinian Authority isolate (Abou Ghanem-­Sabanadzovic et al., 2005).

No symptoms were observed upon mechanical inoculation of the following herbaceous species: Nicotiana benthamiana, N. tabacum, N clevandii, Chenopodium murale, C quinoa, C. amaranticolor, and C. foetidum (Gentit et al., 2001a), indicating that these are either symptomless hosts or non-­host species.

There are currently no other known herbaceous hosts for ApLV, but the efforts to identify other ones have been very limited.

Transmission. No vector is currently known for any of the ApLV isolates or variants. The virus can be transmitted mechanically to herbaceous hosts and by grafting to woody hosts.

Seed transmission is likely absent: when seeds from ApLV-­ infected and clearly symptomatic GF-­305 plants were planted and seedlings observed during a 2-­year period, no signs of infection were recorded (Zemcic, 2006).

Geographical Distribution and Epidemiology ApLV was originally isolated in Moldova from the Bulgarian apricot cultivar ‘Silistra 4.’ This cultivar was introduced to Moldova because of its well-­k nown resistance to many plant pathogens (Zemcic et al., 1998). To our knowledge, there is no further information available on the spread of ApLV in Moldovian commercial orchards. The Silistra province of Bulgaria, the home of the Apricot Research Station, is located in northeastern Bulgaria. More than 600 cultivars and clones have been introduced there since 1953 from almost all the countries where apricots are commercially grown (Tzonev et al. 2000). The PAS variant of ApLV was detected in France in apricot variety ‘Bebeco’ introduced there from Greece (Gentit et al., 2001a, 2001b). Originally the PAS disease was described in the United States, but no sequence of American PAS isolates are currently available, with the possibility that the symptoms observed in the United States may have been caused by a different agent. The status of ApLV in the United States therefore remains in doubt. The PSRS sequenced isolate was derived from the Italian apricot cultivar Caserta 12, grown in Caserta, Southern Italy. PSRV was recorded earlier in the Lecce region of Italy (Grasseau et al., 1999). ApLV was detected in apricot cv. ‘Mistikawi’ in Palestine Authority and in the Van region of Eastern Anatolia, Turkey (Sipahioglu et al., 2006; Usta et al., 2007). Overall, these studies show the presence of ApLV in several countries of the Mediterranean basin or of central Europe but given its mostly latent nature in apricot, ApLV is likely to have been overlooked in many situations, so its distribution is likely to be substantially larger than currently documented.

Detection ApLV can be detected by biological indexing and by serological or molecular assays. The GF305 peach seedling is a woody indicator of choice for the detection of ApLV and related

viruses. N. occidentalis B37 and N. occidentalis ssp. oblique (Zemcic et al., 1993, Gentit et al., 2001a) can be used as herbaceous indicators, but the sensitivity of herbaceous indexing is very poor. Since ASPV is not known to infect stone fruit trees but shows serological cross-­reactions to ApLV, the use of polyclonal antisera directed against ASPV in western blotting assays or immune electron microscopy may be useful as a first step toward identification of ApLV. Recently, F(ab)2 fragments were successfully isolated from an antiserum against ApLV that reacted specifically with ApLV in indirect ELISA assays but not with ASPV (Zemcic, 2006).

Several molecular tools are currently available to identify ApLV-­related sequences in plant tissues and constitute the best strategy for the detection of ApLV (Fig. 19.8) (Nemchinov et al. 1995, 2000). These RT-­PCR assays can be performed with any of three pairs of ApLV-­specific primers derived from the ApLV CP: • Primer pair H-­ALV1/C-­ALV-­1, targeting the region of the ApLV CP with high similarity to ASPV ORF5 (product size 200 bp) • Primer pair H-­ A LV2/C-­ A LV2, targeting the more specific part of the ApLV CP sequence (product size 242 bp) • Primer pair H-­ALV3/C-­ALV3, targeting a fragment corresponding to both the ApLV-­specific and ASPV-­related regions of the ApLV CP (product size 491 bp) The combination of these 3 sets of primers would lead to the identification of genuine ApLV sequences in target tissues.

These primers have been used with success to identify ApLV variants in the Palestine Authority (Abbadi et al., 2003; Abou Ghanem-­Sabanadzovic et al., 2005) and Turkey (Sipahioglu et al., 2006; Usta et al., 2007).

In addition, two cRNA probes have been synthesized either from the first 873 nucleotides of the available 1,444 nt-­long ApLV sequence (Nemchinov and Hadidi, 1998) or from the ASPV-­related portion of the ApLV CP (cDNA clone obtained from the PCR product amplified with primers H-­ ALV1/C-­ ALV1, Nemchinov et al., 2000). The corresponding probes have been used successfully to identify the PAS and PSRS isolates in apricot and peach tissues (Gentit et al., 2001b) and to confirm the presence of ApLV in the Palestine Authority (Abou Ghanem-­Sabanadzovic et al., 2005).

Control Visual assessments are insufficient to locate infected trees due to the general asymptomatic nature of the disease in apricot, but can potentially be a fast and easy way to prediagnose ApLV, PSRSV, or PASV infection on peach cultivars at the end of the growing season. Since diagnostic methods to identify ApLV and related viruses are currently available, a primary control measure is the use by growers of certified virus-­free planting material.

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