Distribution of Tobacco Rattle Virus in Tubers of Resistant and Susceptible
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Amer J of Potato Res (1999) 76:191-197 191 Distribution of Tobacco Rattle Wlrus in Tubers of Resistant and Susceptible Potatoes and Systemic Movement of Virus into Daughter Plants James M. Crosslin, Peter E. Thomas*, and Charles R. Brown USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350. *Corresponding author. Peter E. Thomas; ph. (509) 786-9249; fax (509) 786-9277; email: [email protected]. ABSTRACT Ploeg eta/., 1992). In potato (Solanum tuberosum L.), TRV incites a disease called spralng or corky ringspot (CRS), char- Fifty-nine potato cultivars or breeding clones were acterized by internal discoloration which may form arcs or planted near Umatilla, OR and/or Pasco, WA, in fields circles. The affected tissue may have a corky texture and known to be infested with tobacco rattle virus (TRV) symptoms may be visible on the tuber surface (Harrison and and vector nematodes, Paratrichodorus allius Jen. Robinson, 1986). The virus may be present in asymptomatic (Sid.). Tubers from these field plots were cut and tissues of CRS-affected tubers of at least one cultivar (Cross- examined for corky ringspot (CRS) symptoms. Reverse lin and Thomas, 1995). transcription-polymerase chain reaction (RT-PCR) for Potatoes grown from TRV-infected tubers may become TRV was conducted on tissue samples from sympto- systemically infected and occasionally produce "stem mot- matic and asymptomatic tubers. Sixty-five percent of tle" symptoms where one or a few stems exhibit varying the symptomatic and 42% of the asymptomatic tissue degrees of mottling and discoloration. Systemic movement samples from CRS symptomatic tubers contained of TRV, however, occurs infrequently (Oswald and Bowman, detectable TRV. Approximately 2% of plants grown 1958; Harrison and Robinson, 1982; Harrison and Robinson, from either symptomatic or asymptomatic tubers con- 1986) and may relate to the level of CRS resistance of a par- tained TRV when tested by ELISA, whereas 20% and ticular cultivar (Harrison and Cooper, 1974). 12% of plants grown from symptomatic and asympto- The research described herein was conducted in order matic tubers, respectively, were positive for TRY by to (1) investigate the distribution of TRV in tissue of CRS RT-PCR. These results suggest that RT-PCR is a more symptomatic and asymptomatic tubers of numerous culti- sensitive assay for detection of TRY. Systemic infec- vars and breeding clones, and (2) determine the frequency tions by TRV were detected more often in foliage of of TRV movement from seed pieces into daughter plants. CRS-susceptible genotypes. Daughter tubers exhibit- ing symptoms of CRS, and which contained RT-PCR- MATERIALS AND METHODS detectable TRV, were produced on plants of three genotypes, including one from an asymptomatic parent CRS Evaluation tuber. in 1996, tubers of 28 potato cultivars or breeding clones were planted near Umatilla, OR in an area where CRS had INTRODUCTION been previously observed. Similarly, 31 cultivars or breeding clones were planted both at the UmatiUa site and another site Tobacco rattle virus (TRV) is a bipartite rod-shaped near Pasco, WA where a high incidence of CRS had been virus with a positive sense RNA genome and is transmitted in observed (unpublished data). The materials and sites which a persistent manner by several species of Paratrichodorus were planted are shown in Table 1. Six rephcates of 10 plants and T~chodorus nematodes (Harrison and Robinson, 1986; were planted in randomized blocks. Twenty tubers of each replicate were harvested and 10 tubers were quartered and evaluated for CRS symptoms. The mean percentage of tubers showing CRS symptoms was used as an indicator of CRS Accepted for publication December 14, 1998. resistance. The remainder of the tubers were kept in storage ADDITIONAL KEY WORDS: RT-PCR, ELISA, virus detection, Solanum and used in additional experiments described below. De- tuberosum. 192 AMERICAN JOURNAL OF POTATO RESEARCH Vol. 76 TABLE 1.--Potato c~dtivars or brveding dones grown in two Seed Pieces Grown for Individual Genotype Site a tested by PCRb Tuberization~ Plant PCRd anky ringspot-aff~ed fields in 1996 and subsequent TX1385.12RU U U evaluation of the tube~ and daughter plants for TXAV657.27 U U tobaoco rattle virus. TXNSll2 U Seed Pieces Grown for Individual TXNS278 U U Genotype Site a tested by PCRb Tuberization~ Plant PCR ~ White Rose U,P U,P Yukon Gold U A082706.2R U -~ Russet Burbank~ Control yes yes yes A085165.1 U U U A087119.3 U U U U asix replications of 10 plants each per genotype were planted at the indi- A77715.6 U,P U,P cated sites. U: Umatilla, OR; P: Pasco, WA. Tubers were cut and examined A81480.6 U for corky ringspot symptoms. A8259.3 U,P bTubers produced at the indicated site(s) were tested by RT-PCR for TRV. A8259.5 U,P U,P P P c5-6 plants were placed into growth chambers with 12 hr photoperiod for A8292.5 U,P tuberization. A8390.3 U,P U,P P P dLeaves from individual plants within the set of 10 greenhouse grown A84118.3 U plants were tested for TRV by RT-PCR. A86102.6 U eNot tested. A87109.10 U fCertified Russet Burbank tubers which served as negative controls. A8787.2 U,P P P A8792.1 U A8793.6 U,P U,P A88345.2 U tailed procedures and results of the CRS evaluations will be A88356.1 U published elsewhere. A8836.5 U A88431.1 U A8894.8 U Reverse Transcription-Polymerase Chain A89875.5 U,P U,P U U Reaction (RT-PCR) of Tuber Tissue Abnaki U,P AC87313.3 U Tubers from 35 of the field plots (Table 1) were cut and Achirana U,P examined for CRS symptoms. Two core samples of approx- Atlantic U,P U U imately 7 mm diameter by 6 mm were excised from the ATX84706.2 U,U U AWN85510-2 U,P U,P tubers, combined, placed into microcentrifuge tubes, and BCO894.2 U stored at -20 C. Tissue samples consisted of one or more of Belrus U,P the following: CRS symptomatic tissue from symptomatic Bintje U,P U,P Bzura U,P tubers, asymptomatic tissue from symptomatic tubers and Chipeta U,P P P asymptomatic tissue from asymptomatic tubers. Tissue from Cisa U,P U,P disease-free certified seed pieces of Russet Burbank served CO85026.4 U U as negative controls and tobacco (cv. Samsun NN) tissue sys- CO86142.3 U CO86218.2 U temically infected with TRV served as positive controls. CO87106.5 U Tissues were thawed and ground with a mortar and pes- Dark Red Norland U,P fie. Total nucleic acids were isolated using the procedures of Elba U,P Kennebec U,P U,P U U Presting et al. (1995), resnspended in 500 ~ of sterile distilled Lemhi U,P P P water, and stored at -20 C. A total of 220 tuber samples were N42-11 U processed. RT-PCR for TRV was performed as previously Nemarus U,P Norkotah Russet U,P U,P described (Crosslin and Thomas, 1995). Ten microliters of NZA8904-2 U,P U,P the RT-PCR reaction were resolved on a 1.5% agarose gel Ontario U,P containing ethidium bromide. Samples were considered pos- Pirola U,P itive if a band of the predicted size (463 base pairs) was visi- Pungo U,P Ranger Russet U,P P P P ble when the gel was observed under UV light. Red La Soda U U U Russet Burbank U,P U,P U,P U,P Systemic Movement of TRVfrom Seed Pieces Sangre U Serrana U,P U,P Tubers from 90 of the Umatilla and Pasco field plots 1999 CROSSLIN, eta/.: TOBACCO RATILE VIRUS 193 were cut and examined for CRS symptoms in March 1997. susceptible to CRS included Norkotah Russet, Ranger Rus- Tuber pieces containing a single eye were planted into com- set, Kennebec, Dark Red Norland, and Lemhi. Together with mercial potting mix (Sunshine #1, Sun Gro Horticulture, Inc., Russet BLlrbank these include the most economically impor- Bellevue, WA) in a greenhouse with a 16 hr photoperiod and tant cultivars grown in the Pacific northwest. Most cultivars temperatures of approximately 22-30 C. Ideally, six samples or breeding clones which were grown at both locations had from CRS-symptomatic and four CRS-asymptomatic tubers a higher disease incidence at the Pasco site. For discussion were planted. For some cultivars or clones, however, six purposes, resistant, intermediate, and susceptible lines were symptomatic tubers were not found and symptomless tubers defmed as exhibiting 10% or less, 10.1-19.9%, and 20.0% or were substituted as necessary. The same tubers sampled for greater incidence of CRS, respectively, in either or both of RT-PCR were included in the planting. A total of 294 and 150 the 1996 Umatilla and Pasco plots. The specific data on the symptomatic and 296 and 160 asymptomatic tubers were CRS evaluation of these cultivars and breeding clones will planted from UmatiUa and Pasco, respectively. be published elsewhere. Approximately 2 weeks after emergence, individual leaves were removed and assayed by indirect enzyme-linked RT-PCR of Tuber Tissue immunosorbent assay (ELISA). (Converse and Martin, 1990) Tobacco rattle virus was detected by RT-PCR in 65 % for TRV using antiserum PVAS 820 from the American Type (46/71) of the CRS-symptomatic tissue samples excised from Culture Collection as detecting antibody. Tests were consid- tubers of 18 of the 20 genotypes tested (Table 2). Forty-two ered positive if the absorbance at 405 nm was greater than percent (35/83) of the asymptomatic tissue samples from two times that of healthy potato foliage.