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Peer Reviewed

2005 Plant Management Network.
Accepted for publication 11 July 2005. Published 27 July 2005.

Expansion of the Host Range of Impatiens necrotic spot virus to Peppers

Rayapati A. Naidu, Irrigated Agriculture Research and Extension Center, Department of Plant Pathology, Washington State University, Prosser, WA 99350; and Carl M. Deom and John L. Sherwood, Department of Plant Pathology, The University of Georgia, Athens, GA 30605

Corresponding author: Rayapati A. Naidu.

Naidu, R. A., Deom, C. M., Sherwood, J. L. 2005. Expansion of the host range of Impatiens necrotic spot virus to peppers. Online. Plant Health Progress doi:10.1094/PHP-2005-0727-01-HN.

During spring and summer of 2004, pepper (Capsicum annuum L. cv. Dempsey) plants grown under research greenhouse settings showed discontinuous and irregular necrotic or blackened streaks on stems and petioles (Fig. 1). Leaves showed water-soaked lesions that expanded with time into irregular-shaped necrotic patches. Subsequently, symptomatic leaves exhibited mild yellowing and necrosis along the veins and petiole, and eventually dropped from the plant (Fig. 2). When young plants became infected, they were severely stunted and produced very few fruits. The fruits on infected plants showed uneven ripening with necrotic and concentric green rings (Fig. 3).


Fig. 1. Pepper plant naturally infected with INSV in a greenhouse setting showing extensive necrosis of stem and petioles. Necrosis and "scorching" of lamina is also seen in some leaves.



Fig. 2. Pepper leaves from plant naturally-infected with INSV showing initial symptoms of irregular necrotic lesions on (A) abaxial and (B) adaxial surface. These leaves subsequently showed (not shown in the picture) mild yellowing and necrosis of veins and petiole.



Fig. 3. Effect of natural infection of INSV on pepper fruits. Pepper fruits from infected plants show (A) discontinuous necrotic rings and (B) uneven ripening and green concentric rings. Some fruits also show breaking near the fruit stalk.


Symptomatic leaves were initially tested for Tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) (1) using a DAS-ELISA kit (Agdia Inc., Elkhart, IN), and were negative for TSWV infection. However, subsequent testing of these plants with a DAS-ELISA kit for Impatiens necrotic spot virus (INSV) suggested that the diseased plants were infected with other tospovirus species. Additionally, the skin of fruits from infected plants tested positive for INSV. To confirm the presence of INSV, total RNA was extracted from symptomatic pepper leaves using a RNeasy plant minikit (Qiagen, Inc., Valencia, CA) and used for reverse transcription-polymerase chain reaction (RT-PCR) amplification of the nonstructural protein (NSs) gene of INSV. The sequences of the forward and reverse primers used for RT-PCR were 5-ATGTCTAGTGCAATGTATGAAAC-3 (identical to nt 63-85 of INSV S-RNA, Gene Bank Accession no. NC_003624) and 5- GTTAGTTTAAATCTAATTTAG-3 (complementary to nt 1393-1415), respectively. These primers directed the amplification of a PCR product of about 1300 nucleotides from total RNA from symptomatic leaves (Fig. 4). No product was amplified from total RNA from leaves of non-symptomatic plants. To verify that the amplified products were derived from INSV RNA, the PCR fragment was cloned into pGEM-T Easy vector (Promega, Madison, WI) and two independent clones were sequenced in both directions. Sequence analyses of the cloned PCR product showed 98.8 percent nucleotide sequence identity, and 98.4 and 99.1 percent amino acid identity and similarity, respectively, with a previously published INSV-NSs sequence (Gene Bank Accession no. NC_003624). These results further supported the identity of INSV in symptomatic pepper plants.


Fig. 4. RT-PCR analysis of total RNA extracted from pepper leaves of healthy plants (Lane 2) and those inoculated with INSV by thrips transmission (Lane 3). A DNA fragment of approximately 1.3 kilobase pairs (kpb) was amplified only from total RNA extracted from thrips-inoculated plants, which developed symptoms similar to those in Figs. 1 and 2. RT-PCR of total RNA extracted from E. sonchifolia [(L.) DC. Ex Wight] inoculated with sap prepared from INSV-infected or mock inoculated pepper plants are shown in Lanes 4 and 5, respectively. DNA molecular weight markers (1 kb ladder, Invitrogen, CA) are shown in Lane 1. The sizes (in kbp) of marker fragments are shown on the left.


Additionally, thrips transmission experiments were conducted (3) using symptomatic leaves from diseased peppers as a source for 24 h acquisition access by synchronous-aged (4-h-old) first-instar larvae (~200 per experiment) of western flower thrips (WFT; Frankliniella occidentalis, Pergande.) that had been reared on green bean pods in the laboratory (22 2C under constant light) and not exposed to INSV. After the acquisition access, the larvae were transferred from symptomatic leaves to green bean pods and reared to adults. These potentially viruliferous adult thrips were given a 24-h inoculation access to one month old seedlings of pepper cv. Dempsey (10 thrips per plant and 10 plants per experiment). Three independent experiments were conducted using symptomatic leaves from different plants for virus acquisition. Inoculated plants were maintained in a growth chamber (25 2C, 16-h photoperiod) for symptom development. One week to ten days after thrips inoculation, all plants developed symptoms as shown in Figs. 1 and 2. The presence of INSV in thrips-inoculated plants was confirmed by RT-PCR and DAS-ELISA kit (Agdia Inc., Elkhart, IN) as described above. Finally, we were also able to sap-transmit INSV from symptomatic peppers to the indicator plant Emilia sonchifolia [(L.) DC. Ex Wight] and confirm infection of the indicator by RT-PCR with the INSV primer pair (Fig. 4). Together, these results indicate the host range of INSV includes pepper.

Although this study reports the occurrence of INSV on pepper under greenhouse conditions, its incidence and severity in pepper under field conditions is yet to be realized. TSWV has been pandemic in the United States since the 1980s in both agronomic and horticultural crops. When INSV first emerged in floriculture and nursery crops, it was thought INSV had a restricted host range compared to TSWV (2). However, in recent years, INSV has been detected in other crops like peanut, tobacco, and potato as well as several weed species (4). Because INSV is vectored by WFT and tobacco thrips (F. fusca Hinds.) (3), its expanding host range could make it an economically important problem in agricultural and horticultural crops in the United States.

Literature Cited

1. Adkins, S. 2003. Tomato spotted wilt. Pages 39-40 in: Compendium of Pepper Diseases. K. L. Pernezny, P. D. Roberts, J. F. Murphy, and N. P. Goldberg, eds. American Phytopathological Society Press, St. Paul, MN.

2. Daughtrey, M. L., Jones, R. K., Moyer, J. W., Daub, M. E., and Baker, J. R. 1997. Tospoviruses strike the greenhouse industry: INSV has become a major pathogen of flower crops. Plant Dis. 81:1220-1230.

3. Naidu, R. A., Deom, C. M., and Sherwood, J. L. 2001. First report of Frankliniella fusca as a vector of Impatiens necrotic spot tospovirus. Plant Dis. 85:1211.

4. Perry, K. L., Miller, L., and Williams, L. 2005. Impatiens necrotic spot virus in greenhouse-grown potatoes in New York state. Plant Dis. 89:340.