VOLUME 8 NUMBER 1 MARCH 1979

RESEARCH NOTES

Avocado Sunblotch - A Viroid Disease?Wayne Thomas and N.A. Mohamed

Plant Disease Division,Department of Scientific and Industrial Research,

Private Bag, Auckland, New Zealand.

Sunblotch is a serious disease affecting avocados inCalifornia (7), Florida (16), Peru (18), Israel (1), SouthAfrica (9) and Venezuela (11), though it is not known to oc­cur in New Zealand plantings. The infectious nature of thedisease has been established by grafting (7) and seedtransmission experiments (17) but the causal agent has notbeen characterized. The disease occurs in two forms(a) A disease where characteristic symptoms are dis­

played - yellow streaking and spotting of the bark oftWigs and young branches, and irregular white toyellow or reddish yellow sunken areas on the fruit.Only a small percentage (2-5%) of fruits from suchtrees show the fruit symptoms, and only those fewfruits with symptoms have seeds that carry the disease.

(b) A symptomless disease, in which the plant carries theinfection without displaying any of the known symp­toms of the disease. Most (86-100%) of the fruits fromthese plants carry the disease (17). Seedlings fromsuch trees remain symptomless but infect and maycause symptoms to develop in scions grafted on tothem. Use of such seed as rootstock represents themain mode of spread for the disease (17).

Sunblotch was originally thought to be caused by a virusbecause it was graft transmissible; no virus particles havebeen found by electron microscopy of extracts or in sec­tions from infected tissue, nor has it been possible totransmit the disease mechanically or with insects. We havetherefore investigated the possibility that the disease maybe caused by a viroid. We report the presence of a lowmolecular weight RNA species in extracts from leaves ofsymptomless carrier trees and also in extracts fromsymptom-bearing bark stripped from infected trees.Similar low molecular weight RNA species (viroids) havebeen found to cause the diseases potato spindle tuber (6),citrus exocortis (14), chrysanthemum stunt (5), andcadang-cadang of coconuts (12). We conclude that thecausal agent of sun blotch in avocado is also likely to be aviroid.

For our studies symptomless and symptom-bearingplants were raised from seed, from known infected symp­tomless plants, obtained from Dr J. M. Wallace, Universityof California, Riverside. We first searched for unusualnucleic acid species in leaves from symptomless,symptom-bearing and healthy trees. Total leaf nucleicacids were extracted by phenol-SDS (4) or by chloroform­butanol followed by phenol (10). Nucleic acids wereprecipitated with ethanol, resuspended in distilled water,dialysed against distilled water, reprecipitated with ethanoland finally resuspended in 0.04 M tris acetate pH 7.5 +0.002 M Na acetate containing 0.2% SDS. Samples werelayered onto 2.5% polyacrylamide gels (P.A.G.): elec­trophoresed for 2-3 hours (Fig. 1), and stained withtoluidine blue. Ribosomal RNA, DNA, 4S and 5S RNA weredetected in all samples. Samples from symptomlesscarrier tree leaves (cv, Topa Tepa) contained an additionalnucleic acid of mobility equivalent to about 8S that was notpresent in extracts of leaves from healthy or symptombearing trees. Subsequent experiments demonstrated thepresence of the 8S nucleic acid in two additional knownsymptomless carrier trees of the same cultivar, originallyimported from Riverside, California as infected symptom­less carrier seed. The 8S nucleic acid was not present inhealthy Topa Topa or Hass trees. To provide a further test,leaves of a symptomless carrier tree were imported fromSouth Africa (through the courtesy of Dr J. V. Da Graca,Citrus and Subtropical Research Institute, Nelspruit, SouthAfrica) and the 8S nucleic acid was found again in theseleaves. It thus appears that this 8S nucleic acid is presentin leaves of symptomless trees but not in those of trees withsymptoms. Could this be because the symptomsthemselves were a type of hypersensitive reaction? If so,the nucleic acid might be confined to the areas showing thesymptoms. We therefore stripped the bark from stemsshowing marked sun blotch symptoms and extractednucleic acids from it and also from the underlying wood.No 8S nucleic acid was found in extracts from the wood butit was detected in the bark samples, though at a lower con­centration than that found in symptomless tree leaves. Weconclude that the 8S nucleic acid represents the infectiveagent of sunblotch.

When the nucleic acid extract in SSC buffer (0.15 MNaCI + 0.015 M Na citrate pH 7.0) was treated with 1pg/ml of ribonuclease A, the leaf ribosomal RNA wasdigested while the suspected viroid RNA remained intact:at 10 pg/ml of ribonuclease A the suspected viroid RNA

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was also digested and could not be detected by PAG.electrophoresis. On treatment of the nucleic acid extractwith DNAase at 10 .ug/ml in 0.1 SSC + 1 mM MgCI 2buffer,the leaf DNA was digested but ribosomal RNA and 8Snucleic acid were still present. These results indicate thatthe 8S nucleic acid is an RNA having a fairly complexsecondary structure similar to that of the known viroids (4).

sun blotch nucleic acid . Although some of the knownviroids (CEV and PSTV) are easily transmitted mechanical­ly to a small and specific host range, at least one other, thatof cadang-cadang was not transmitted until well after itsviroid nature had been established, and then only withsome difficulty (13).

The occurrence of sunblotch nucleic acid in leaves ofsymptomless carrier trees in high concentrations, in barklesions of trees with stem symptoms and its absence inleaves and wood of trees with stem symptoms, suggeststhat in trees bearing such symptoms the infectious nucleicacid is localized in the phloem in the bark and in some fruit(2-5%), being confined in such areas because of thehypersensitive reaction of susceptible seedlings. In symp­tomless carrier trees, however, there is no hypersensitivereaction and no localization of the sun blotch nucleic acid,and so the nucleic acid is found readily in the leaves. Thiswould explain why the rate of seed transmission is so highin symptomless carrier trees (86-100%) and low in treeswith stem and fruit symptoms (less than 5%).

These findings are of considerable importance at thepractical level. Until now, sym pt omless carrie rs ofsunb!otch disease could be detected only by a laboriousgraft indexing procedure (Dr J. M. Wallace , personal com­muni cation), requiring considerable effort and a mini mumperiod of two years. Detection of sunblotch RNA in extractsfrom leaves of symptomless carrier trees is much simpler,and requires a period of only 4 days. This should greatlyfacilitate disease surveys, as well as other types ofresearch on sunblotch in avocado.

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REFERENCES

Fig. 1. Polyacrylamide gel electrophoresis of total nucleic acidsfrom (a) E. coli; (b) avocado symptomless carrier leaves; (c) leavesof known healthy avocado; (d) stem material from symptom bear­ing avocado; (e) leaves from symptom-bearing avocado. Thenucleic acids were layered on 2.5% polyacrylamide - 0.5%agarose gels and electrophoresed for 2.5 h at 4 watts (constantpower). The electrophoresis buffer used was Trisphosphate ­EDTA containing 0.2% SDS (8). Note the presence of the extra 8Snucleic acid in the symptomless carrier (b) which was not presentin the healthy control (c) or the symptom bearing trees (d and e).

(1) Comelli. A. (1960)- Subtropical fru it cu lt ivation in Israel. IV.The avocado in Israel. Fruit D'outre lV1er 15: 261-274.

(2) Diener, T. O. (1971) - Potato spind le tuber 'virus'. IV. Areplicating, low molecular weight RNA. Virology 45: 411­428.

(3) Diener, T. O. (1974) - Viroids: the smallest known agents ofinfectious disease. Annual Review of Microbiology 28: 23­89.

(4) Diener, T. 0., Hadidi, A., and Owens, R. A. (1977) ­Methods for studying viroids. Methods in Virology 6: 185­217.

(5) Diener, T. 0., and Lawson, R. H. (1973) - Chrysanthemumstunt: a viroid disease. Virology 51: 94-101.

(6) Diener, T. 0., and Raymer, W. B. (1967) - Potato spindletuber virus: a plant virus with properties of a free nucleicacid. Science 158: 378-381.

(7) Horne, W. T., and Parker, E. R. (1931) - The avocado dis­ease called sun blotch. Phytopathology 21: 235-238.

(8) Loening, U.E. (1968)- Molecular weights of ribosomal RNAin relation to evolution. Journal of Molecular Biology 38:355-365.

(9) Loest, F. C., and Stofberg, F. J. (1959) - Avocado diseases.Farming in South Africa 29: 517-520.

(10) Morris, T. J., and Wright, N. S. (1975) - Detection onpolyacrylamide gel of a diagnostic nucleic acid from tissueinfected with potato spindle tuber viroid. American PotatoJournal 52: 57-63.

(11) Rondon, A., and Figueroa, M. (1976) - Sun blotch ofavocado Persea americana in Venezuela. AgronomicaTropical 26: 463-466.

(12) Randles, J. W. (1975) - Association of two ribonucleic acidspecies with cadang-cadang disease of coconut palm.Phytopathology 65: 163-167.

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To determine the molecular weight of 8S RNA, total leafnucleic acids were submitted to P.A.G. electrophoresis un­der non-denaturing conditions. The sun blotch RNA wascalculated to have a molecular weight of 60-70,000 using E.coli 23S and 16S ribosomal RNA (1,080,000 and 560,000)and avocado 4S and 5S RNA (26,000 and 36,000) asmarkers (2). This compares with molecular weights of50,000 for potato spindle tuber (3), 50-60,000 for citrusexocortis (15), and 84,000 for cadang-cadang (12) nucleicacids under non-denaturing conditions. From the aboveevidence it appears that the 8S nucleic acid is the infectiveagent of sunblotch.

Symptoms of avocado sunblotch take 3 to 9 months toappear when infected material is grafted to healthyseedlings. Because of this time lag before symptomsappear, we have not yet been able to establish thepathogenicity of the sun blotch nucleic acid, but ex­periments are in progress to infect healthy avocadoseedlings with nucleic acid extracts containing the

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(13) Randles, J. W., Boccardo, G., Retuerma, M. L., and Rillo, E.P. (1977) - Transmission of the RNA species associatedwith cadang-cadang of coconut palm, and the insensitivityof the disease to antibiotics. Phytopathology 67: 1211­1216.

(14) Semancik, J. S., and Weathers, L. G. (1972) - Exocortisvirus: an infectious free-nucleic acid plant virus with un­usual properties. Virology 47: 456-466.

(15) Semancik, J. S., and Weathers, L. G. (1972) - Exocortis dis­ease: evidence for a new species of 'infect ious' lowmolecular weight RNA in plants. Nature New Biology 237:242-244.

(16) Stevens, H. E. (1939 ) - Avocado sunblotch in Florida.Phytopathology 29: 537-541.

(17) Wallace, J. M. , and Drake, R. J . (1962) - A high rate of seedtransmission of avocado sunblotch virus from symp­tomless trees and the origin of such trees. Phytopathology52: 237-241.

(18) Zentmyer, G. A. (1959) - Avocado diseases in LatinAmerica. Plant Disease Reporter 43: 1229.

Avocado Affected By Sunblotch DiseaseContains Low Molecular WeightRibonucleic Acid

J. L. Dale and R. N. AllenPlant Pathology Branch , Dept. of Primary Industries,

Indooroopilly, ald. 4068 .

Sunblotch disease of avocado (Persea americana) wasfirst described in California over 50 years ago (3) and morerecently in Australia (6). Its cause is unknown though , as itwas shown to be transmitted by grafting and through seed,control of the disease using scion and seed certificationschemes has been successful. Graft transmission mayresult in severe symptoms after three months incubationbut usually the incubation period is 12 months or longer.Seed transmission from symptomless trees is commonand invariably leads to systemic infection of the plant butwithout symptoms being displayed (7).

The detection of symptomless carriers of the disease is amajor concern to avocado growers. Detection sometimesoccurs accidentally in nurseries, when healthy susceptiblescions are grafted onto systemically infected rootstocks,but more commonly it is only found by systematic testing(8). Apart from the disease agent being transmittednaturally through root grafts (8) and seed (7), no othernatural means of transmission is known.

As no virus-like particles have been seen in sections orextracts of diseased tissue, we decided to examine dis­eased tissue for unusual ribonucleic acid (RNA) speciessimilar to those associated with citrus exocortis and po­tato spindle tuber diseases (2, 5). The work was further en­couraged by the recent observation that sunblotch wastransmitted on knives and pruning implements at a smallbut erratic frequency (4).

We examined two isolates of sunblotch disease; anAustralian isolate (6) and an isolate obtained from graftinga healthy Hass avocado seedling with symptomlessly in­fected Todd avocado imported from the University ofCalifornia, Riverside, U.S.A. These isolates have beenmainta ined at Wollongbar, N.S.W. for indexing purposes.

Infected and healthy avocado tissue was extracted us­ing a mod ification of the method described by Semancikand Weathers (5) . Extracts were analysed by elec­trophoresis in 5% polyacrylamide gels at 5 mA/gel for 4hours in a tris-acetate-EDTA buffer system (1). Gels werestained in 0.2% toluidine blue in acetate buffer anddestained in water to detect RNA bands.

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RNA species from healthy Hass avocado and avocadoinfected with the Californian sunblotch isolate are shown infigure 1. A band we call SB-RNA with an Rf of 0.57 (com­pared to the bromophenol blue ma rke r) was present in allpreparations from infected avocado but absent frompreparat ions of healthy avocado, eve n when gels wereloaded with up to six times the concentration needed to seeSB-RNA in preparations from infected material. SB-RNAwas detected in preparations from young shoots, leavesand mature stems. Both the Californian Todd andAustralian isolates contained SB-RNA. Extracts from as lit­tle as four grams of infected tissue gave an obvious SB­RNA band. Digesting the extract with bovine pancreaticribonuclease (20,ug/ml for 15 min . at 20°C) eliminated theSB-RNA band as well as most, but not all , of the otherbands evident in figure 1.

Figure 1. Separation of nucleic acid species by electrophoresis in5% polyacrylamide gels . Extracts from healthy (left) and sunblotchinfected (r ight) Hass avocado plants. The arrow indicates the posi­tion of the SB-RNA band.

The position of the SB-RNA band in gels is close to thatof potato spindle tuber viroid and other viro ids elec­trophoresed under the same conditions (2, 5), indicatingthat the SB-RNA has a molecular weight similar to that ofviroids.

The evidence presented here strongly suggests thatavocado sun blotch is caused by a viroid. Work is inprogress to investigate the physio-chemical cha racteristicsof SB-RNA and establish its pathogenicity, host range andrelationship to known viroids.

It will be much simpler to test avocado material forsun blotch by analysing its RNA for SB-RNA than the costlyand time consuming method of grafting usually employed.Th is should enable epidemiological studies in the field tobe done on a scale not previously possible.