Proc. Fla. State Hort. Soc. 123:5–7. 2010.
Results of the 2009 ASBVd Survey of Avocado Accessions of the National Germplasm Collection in Florida
Cecile L. Tondo*, Raymond J. Schnell, and David N. Kuhn National Germplasm Repository, USDA ARS, 13601 Old Cutler Road, Miami, FL 33158
Additional index words. Persea americana, avocado diseases, avocado sunblotch viroid, germplasm collection management, viroid indexing The presence of avocado sunblotch viroid (ASBVd) infection among the avocado (Persea americana Mill.) accessions in the National Germplasm Repository at Miami (NGR–Miami) was established in previous studies. An ASBVd specific reverse transcription-polymerase chain reaction (RT-PCR) protocol was used to detect the viroid. Surveys performed in 1996 and in 2000 found that the proportion of ASBVd positive accessions remained unchanged at 19% during that time period. The object of the current study was to assess the spread of infection, if any, and the rate and direction of transmission. For this purpose the collection was screened again for ASBVd in 2009. The germplasm collection increased from 403 to 505 trees. Fifty newly infected trees were detected. Forty-eight percent of the newly infected plants were found to be adjacent to previously infected plants, adjacent to plots from which infected plants had been removed, or adjacent to other newly infected plants that are adjacent to previously infected plants or contaminated plots. No pattern in direction of spread was discerned for non-adjacent new infections. The proportion of plants found to be positive for the viroid in the current study is 21%. Fourteen plants previously found to be infected were found to be negative in this survey. The proportion of infected plants (historically and at present) in the current collection is 24%. The increase in ASBVd infections reinforces the importance of establishing back-up collections at different locations. Establishment of collections at the NGR in Hawaii is currently under way.
Avocado sunblotch viroid (ASBVd) is the causal agent of An initial survey of the NGR–Miami collection of P. americana avocado sunblotch disease that affects Persea americana Mill., was performed in 1996 (Ronning et al., 1996). A second survey the common avocado. This disease is of economic importance was completed from samples collected during the 2000 flowering as expression of disease symptoms on the fruit renders the fruit season (Olano et al., 2002). This report describes findings of the unmarketable. Infection not only results in inferior quality fruit, but third survey performed on the collection in 2009. The objectives also in lower yields as well as a decline in tree health. Symptoms of the current survey were to assess the spread of infection, if include yellow or red depressed areas on the fruit surface that any, and the rate and direction of transmission. turn black over time, bleached veins and petioles on the leaf, and rectangular cracking patterns in the bark of older branches (Fig. Materials and Methods 1); however, the trees can remain symptomless for many years. ASBVd is a small, covalently closed, circular, single-stranded Plant material. Tissue samples for RNA extraction were RNA molecule (Palukaitis et al., 1979). The viroid is transmitted collected from all Persea sp. accessions from the germplasm naturally through budding and natural root grafting (Horne et al., collection in the National Germplasm Repository in Miami 1941; Whitsell, 1952), through seed (Wallace and Drake, 1962) and pollen (Desjardins et al., 1979), and is transmitted artificially by mechanical means on pruning equipment and through grafting. A protocol for routine detection of ASBVd that utilizes reverse transcription and the polymerase chain reaction (RT-PCR) was developed to selectively amplify ASBVd from partially purified RNA extracts (Schnell et al., 1997). This protocol is accepted as a diagnostic means for declaring propagating stocks ASBVd free (Dodds et al., 2010). A modification of the RT-PCR assay was developed for use on capillary electrophoresis (CE) genetic analyzers by incorporating fluorescent detection, allowing high throughput capability. Using CE and single-strand conformation polymorphism (SSCP) analysis, it is possible to estimate the num- ber of variants in crude samples extracted from avocado plants (Schnell et al., 2001a). In addition, the longer a tree is infected with the viroid, the greater the number of ASVBd variants it contains (Schnell et al., 2001b). Fig. 1. Fruit and leaves of avocado (Persea americana) showing symptoms of avocado sunblotch viroid, which include yellow or red depressed areas on the fruit surface (A) that turn black over time, bleached veins and petioles on the *Corresponding author; phone: (786) 573-7086; email: [email protected] leaf (B), and rectangular cracking patterns in the bark of older branches.
Proc. Fla. State Hort. Soc. 123: 2010. 5 (NGR–Miami). Unopened flower buds were collected from all Electrophoresis. Detection of amplified product was per- flowering trees, but mature leaves were collected from those trees formed using capillary electrophoresis on an ABI Prism 3730 that did not flower during the 2009–10 flowering season. One Genetic Analyzer (Applied Biosystems, Foster City, CA) using hundred milligrams of plant tissue were weighed and stored at –80 POP7 polymer and default run modules. °C until extraction. A total of 505 Persea sp. trees were sampled. Indexing. All samples with amplification product of 247 Eighty-one plants were resampled for verification purposes in bp were scored as ASBVd positive; those samples that did not 2010. The accession ‘Aycock Red #3’ located in W3-1-08-02 was produce a 247 bp product were considered ASBVd negative. If used as a positive control, as this plant has displayed symptoms the positive control in the 96-well PCR plate failed to amplify, of ASBVd for many years. then the entire plate was repeated using 5 to 9 µL of undiluted RNA extraction. RNA extractions were performed using a RNA. Results were compared to previous surveys and all positive protocol modified from Ainsworth (1994) and described in Olano samples that had been scored as negative in previous surveys were et al. (2002) with some modifications for high throughput, as collected again, and re-assayed to confirm new status. Also, all follows: extraction buffer was added at room temperature and plants previously indexed as ASBVd positive in earlier surveys, homogenization was carried out in a GenoGrinder2000 (SPEX yet failing to test positive in 2009, were collected again, and re- Certiprep, Metuchen, NJ) at 1500 strokes/minute for a duration of assayed for verification.All samples that failed to be confirmed 4 min for flowers, or 6 min for leaves. Twenty-four samples were were assayed a third time using a different primer pair (Semancik processed at a time in 96-well deep well plates, such that samples and Szychowski, 1994). were placed in non-adjacent wells, and each plate included one positive control. The final RNA pellet was resuspended in RNAse Results and DNase free water and stored at –20 °C. Quantification and normalization. RNA samples were The P. americana sp. germplasm collection contained a total quantified using Quant-iT RiboGreen RNA Reagent (Invit- of 280 accessions in 2009. Many of these accessions are repre- rogen, Carlsbad, CA) on a FLX800 Microplate Fluorescence sented by more than one clone, resulting in a total of 505 trees. Reader (Bio-Tek Instruments Inc., Winooski, VT). A standard The number of trees added to the collection since the last survey curve was generated and readings were taken using the gain in 2000, and thus new to this survey, is 144. Forty-two trees died set to 100 ng·µL–1, and 25 ng·µL–1 standards. RNA dilution or were removed from the collection since the last survey in 2000, plates were prepared such that a quantity of 1 µg of RNA in and 22 of these trees were infected with ASBVd. One hundred a volume between 1 and 9 µL could be sampled from each and six trees tested positive for ASBVd in this survey (Table 1). well of the dilution plate by a liquid handling robot (Multi- The proportion of trees assayed as positive for ASBVd in this probe II; Perkin Elmer, Waltham, MA) for use as template study was 21% (106/505).Of the 106 positive trees, 50 are new in cDNA synthesis. infections since the last survey in the year 2000. Fourteen trees cDNA synthesis and amplification. Synthesis of cDNA was found to be infected in previous surveys were found to be nega- carried out using SMART MMLV RT or PowerScript Reverse tive for ASBVd in this survey. Therefore, the number of trees that transcriptase (Clontech Laboratories, Inc., Mountain View, CA) were indexed as positive in this 2009 survey (106), combined following the protocol outlined in the product inserts, except with those indexed as positive in previous surveys (14), is a total that the polymerase was added simultaneously with other PCR of 120 infected trees in the collection, bringing the proportion of reagents. PCR amplification was carried out using Advantage HD infected trees to 23.8% (120/505). Mix (Clontech Laboratories, Inc.) using the protocol outlined in In order to examine the direction of transmission (i.e., to de- the product insert, except the reaction volume was decreased to termine the source of new infections) we looked at proximity of 10 µL. Primers were labeled with a fluorescent dye as in Schnell the newly infected trees to infected trees and contaminated plots et al. (2001) and 2.5 µmol of primer were added to each reac- (plots from which infected plants have been removed). Twenty- tion. All RT-PCR reactions were prepared using a Multiprobe II four of the 50 newly infected trees were found to be adjacent to liquid handling robot. PCR was carried out on a DNA Engine previously infected trees, to contaminated plots, or to other newly tetrad thermal cycler (BioRad, Hercules, CA) using the follow- infected trees that are adjacent to previously infected trees or ing thermal cycling conditions: 40 cycles of 94 °C for 30 s, 55 plots (Table 1). Twenty-six trees were non-adjacent to infected °C for 30 s, 72 °C for 30 s; and a final 7-min, 72 °C extension. trees or contaminated plots; of these, eight trees were adjacent PCR was carried out in 96-well plates and a positive control was to other newly infected trees that were not themselves adjacent included on each plate. to other infected trees or plots.
Table 1. Results of 2009 survey by field for the for the NGR Persea americana sp. germplasm collection. Total no. No. of New infections No. of trees infected Field of trees infected trees (%) Adjacent Non-adjacent Reversion historically and at present W3-1 36 19 (52.8%) 7 1 2 21 W4-1 42 30 (71.4%) 7 1 2 32 WA-2 63 14 (22.2%) 1 6 7 21 WA-2 seedlings 22 17 (77.3%) 0 1 1 18 WB-3 129 7 (5.4%) 5 2 1 8 WB-4 195 16 (8.2%) 4 12 0 16 Miscellaneous 18 3 (16.7%) 0 3 1 4 Total 505 106 (21.0%) 24 26 14 120 (23.8%)
6 Proc. Fla. State Hort. Soc. 123: 2010. Discussion Infection of ASBVd in the P. americana sp. collection at NGR– Miami has increased at a rate of 4.7% in the last 9 years. Strict In the current survey performed on the Avocado National phytosanitary practices have not contained the viroid. Removal Germplasm Collection in 2009, and reported here, it was found of all infected trees may be necessary in order to keep ASBVd that the proportion of trees assayed as positive for ASBVd was from spreading. If all infected trees were destroyed, 20% of the 21% (106/505).Newly infected trees since the last survey in 2000 accessions in the collection will be lost. These accessions could totaled 50. Fourteen trees previously found to be infected were be replaced as new sources become available. The increase in found to be negative for ASBVd in this survey. As these 14 trees ASBVd infections reinforces the importance of establishing back- are considered infected, then the proportion of infected trees is up collections at different locations. Establishment of collections 23.8% (120/505). Infection of ASBVd in the P. americana sp. at the NGR in Hawaii is currently under way. collection at the NGR has increased at a rate of 4.7% in the last 9 years. Literature Cited In order to determine the source of new infections, we looked at proximity of the newly infected trees to infected trees and Ainsworth, C. 1994. Isolation of RNA from floral tissue ofRumex acetosa contaminated plots (plots from which infected plants have been (Sorel). Plant Mol. Biol. Rptr. 12:198–203. removed/died).Twenty-four of the 50 newly infected trees were Desjardins, P.R., R.J. Drake, E.I. Atkins, and B.O. Bergh. 1979. Pollen found to be adjacent (Table 1). In all these cases, root grafting is transmission of avocado sumblotch virus experimentally demonstrated. the most obvious explanation for the transmission of the viroid. Calif. Agr. 33:14015. Dodds, J.A., D. Mathews, M.L. Arpaia, and G.W. Witney. 2010. Recogniz- No pattern could be discerned for the appearance of new infec- ing avocado sun blotch disease. AvoResearch.
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