First Report of Root Rot Caused by Binucleate Rhizoctonia Anastomosis Group F on Musa spp.

A research program was initiated at the University of Georgia in 2003 to identify banana cultivars suitable for production in the coastal and southern areas of the state. During a root disease survey conducted in October 2007 on bananas (Musa spp.) grown at the University of Georgia Bamboo Farm and Coastal Gardens in Savannah, GA, root lesions and root rot were observed on banana cvs. Gold Finger, Kandarian, and Manzano. Root lesions were dark brown to black and irregular in shape, with partial or entire roots affected. Lateral roots and outer layers of cord roots (roots arising from interior layers of the corm) of infected plants were blackened and rotted. Diseased root samples were collected from three plants of each cultivar, surface sterilized with 0.6% sodium hypochlorite, and placed on tannic acid benomyl agar (TABA). Pure cultures of the fungus consistently associated with diseased tissue were obtained by subculturing hyphal tips on TABA. Mycelia of the fungus on potato dextrose agar (PDA) were light to deep brown and the hyphae tended to branch at right angles. A septum was present in each hyphal branch near the point of origin and a slight constriction at the branch was observed. The hyphae of two isolates were stained with 0.6% phenosafranin and 3% KOH and binucleate hyphal cells were observed. On the basis of these morphological features, the isolates appeared to be binucleate Rhizoctonia anamorphs (teleomorph Ceratobasidium Rogers). For molecular identification, the internal transcribed spacer (ITS) regions and the 5.8S gene from rDNA of the isolates were cloned and sequenced (GenBank Accession No. HQ168370). The ITS regions (775 bp) were 100% identical between the two isolates and 99% identical to Ceratobasidium sp. AG-F strain SIR-1 isolated from sweet potato in Japan (GenBank Accession No. AF354085). The anastomosis group of the isolates was confirmed by pairing with strain SIR-1 on PDA. On the basis of morphological and molecular characteristics and the anastomosis assay, the two isolates were identified as a Ceratobasidium sp. AG-F (1-3). Pathogenicity assays were conducted by inoculating banana plants (cv. Golden pillow, synonym = Manzano) grown in pots under greenhouse conditions (25 to 27°C). Twenty wheat seeds infested with each isolate were placed uniformly around each plant at a depth of 10 cm in the soil. The plants were incubated in the greenhouse and the roots were examined 2 months after inoculation. Brown-to-black lesions and root rot, identical to symptoms associated with field banana roots, were observed on all inoculated plants but not on the noninoculated control plants. The fungus was reisolated from affected root samples and the identity was confirmed by morphological and molecular characteristics and the anastomosis assay. To our knowledge, this is the first report of banana root rot caused by binucleate Rhizoctonia anastomosis group F. With the increased interest in producing bananas for food and ornamental purposes, the occurrence of Ceratobasidium root rot on bananas needs to be considered when designing disease management programs and searching for suitable cultivars for banana production. References: (1) L. L. Burpee et al. Mycologia 70:1281, 1978. (2) D. González et al. Mycologia 93:1138, 2001. (3) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN. 1991.

Biological and molecular analyses of the acibenzolar-S-methyl-induced systemic acquired resistance in flue-cured tobacco against Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) is an economically important virus of flue-cured tobacco. Activation of systemic acquired resistance (SAR) by acibenzolar-S-methyl (ASM) in flue-cured tobacco was studied under greenhouse conditions by challenge inoculation with a severe isolate of TSWV. ASM restricted virus replication and movement, and as a result reduced systemic infection. Activation of resistance was observed within 2 days after treatment with ASM and a high level of resistance was observed at 5 days onward. Expression of the pathogenesis-related (PR) protein gene, PR-3, and different classes of PR proteins such as PR-1, PR-3, and PR-5 were detected at 2 days post-ASM treatment which inversely correlated with the reduction in the number of local lesions caused by TSWV. Tobacco plants treated with increased quantities of ASM (0.25, 0.5, 1.0, 2.0, and 4.0 g a.i./7,000 plants) showed increased levels of SAR as indicated by the reduction of both local and systemic infections by TSWV. The highest level of resistance was at 4 g a.i., but this rate of ASM also caused phytotoxicity resulting in temporary foliar spotting and stunting of plants. An inverse correlation between the TSWV reduction and phytotoxicity was observed with the increase of ASM concentration. ASM at the rate of 1 to 2 g a.i./7,000 plants activated a high level of resistance and minimized the phytotoxicity. Use of gibberellic acid in combination with ASM reduced the stunting caused by ASM. Present findings together with previous field experiments demonstrate that ASM is a potential option for management of TSWV in flue-cured tobacco.

A rapid and efficient inoculation method for Tomato spotted wilt tospovirus.

A rapid and efficient method of inoculation for Tomato spotted wilt tospovirus (TSWV) was achieved by applying the inoculum with a device consisting of a spray gun, an atomizer and a CO2-powered sprayer. The inoculum contained infected leaf sap prepared in 0.1M phosphate buffer, pH 7.0, 0.2% sodium sulfite and 0.01 M 2-mercaptoethanol (1g: 10 ml) and 1% each of Celite 545 and Carborundum 320 grit. The spray application of chilled inoculum at the rate of 1.1 ml/plant and at an air pressure of 4.1 bar resulted in systemic infection nearly to a 100% of the tobacco (Nicotiana tabacum) plants inoculated. The inoculation procedure was successfully applied to two other important host species of TSWV, peanut (Arachis hypogaea) and tomato (Lycopersicon esculentum), where 75.0-100% and 72.2-91.6% plants developed systemic infection, respectively. The approach facilitated a much faster inoculation of test plants with TSWV as it was estimated to be about 50 times quicker (depending on the plant species) than the hand inoculation. The procedure is suitable for rapid and simultaneous inoculation of a large number of test plants with TSWV and should facilitate screening of germplasm and breeding lines for virus resistance.

Effect of Transplant Age, Tobacco Cultivar, Acibenzolar-S-Methyl, and Imidacloprid on Tomato Spotted Wilt Infection in Flue-Cured Tobacco.

Tomato spotted wilt virus (TSWV) has become the most serious problem in flue-cured tobacco in Georgia and is a growing problem in other tobacco-growing areas in the United States. The effects of transplant age (6 to 10 weeks), tobacco cultivar (K-326 and NC-71), and preplant applications of acibenzolar-S-methyl (ASM) and the insecticide imidacloprid (IMD) were evaluated on levels of TSWV infection, number of symptomatic plants, and yield in field trials over 4 years. In all 4 years and in four of five trials, treatment of transplants with ASM and IMD resulted in fewer symptomatic plants, smaller areas under the disease progress curve (AUDPC), and higher yields compared with the nontreated controls. There were no consistent effects of transplant age or cultivar on number of symptomatic plants or systemic infections, AUDPC, or yield. Treatment of transplants with ASM and IMD can significantly reduce the number of symptomatic plants in the field and substantially increase yields and value per hectare.

First Report of Black Leaf Spot of Banana Caused by Deightoniella torulosa in Georgia.

Black spots were observed on the leaves of bananas (Musa spp.) grown at the University of Georgia Bamboo Farm and Coastal Gardens in Savannah, GA in November 2007. Symptoms occurred on more than 60 plants, representing 16 of 34 cultivars of bananas investigated. Most lesions were less than 10 mm in diameter and tan to black. However, larger oval lesions more than 20 mm across with black borders and yellow halos also occurred. Lesions were more prevalent on older leaves. On young leaves, lesions first appeared along the leaf margin near the tip of the leaf on one side of the central vein. Lesions expanded to the entire leaf as the disease progressed, but were more prevalent along leaf margins. Thirty-two diseased leaf samples, two from each cultivar, were incubated at 25°C in the dark and conidia were produced on the lesions 2 days after incubation. Pure cultures of the fungus were obtained from five leaf samples by single-spore culturing on potato dextrose agar medium and identified on the basis of morphological characteristics. Conidia on V8 agar are straight or slightly curved, obpyriform to obclavate, and olive to brown with 3 to 13 septa. Conidiophores are brown and swollen at the apex. The fungus was identified as Deightoniella torulosa (Syd.) Ellis on the basis of morphological characteristics described previously (1,2). Pathogenicity studies to fulfill Koch's postulates were conducted on banana cvs. Dwarf Namwah and Dwarf Nino under greenhouse conditions (25 to 27°C). Six plants of each cultivar were used in one experiment and the experiment was repeated one more time. Banana leaves were inoculated by spraying with a suspension of conidia from a pure culture. Symptoms developed as small black lesions on the leaves of both cultivars within 1 week of inoculation. As the disease progressed, some of the small lesions expanded to form larger oval lesions. Symptoms were identical to those on the field samples and were identified as the black spot disease as described on abaca and banana (2). The fungus was reisolated from symptomatic leaves and the identity was confirmed. No symptoms were observed on noninoculated control plants. The black spot disease has been reported in Florida attacking banana and plantain (3). To our knowledge, this is the first description of the presence of the disease on field-grown banana in Georgia. In recent years, increasing efforts have been made in Georgia in the search of banana cultivars suitable of commercial production in the coastal and southern areas of the state. Black spot of banana is an important disease and its occurrence deserves consideration in evaluating banana cultivars and developing disease management approaches for banana production in Georgia. References: (1) M. B. Ellis. Mycol. Pap. No. 66. CAB International Mycological Institute, Wallingford, UK, 1957. (2) R. H. Stover. Banana, Plantain and Abaca Diseases. Commonw. Mycol. Inst., Kew, Surrey, UK, 1972. (3) C. Wehlburg et al. Bull. 11. Fla. Dep. Agric. Consum. Serv. Div. Plant Ind., 1975.

First Report of Iris yellow spot virus in Spiny Sowthistle (Sonchus asper) in the United States.

Iris yellow spot virus (IYSV) is a member of the genus Tospovirus in the family Bunyaviridae. Its known host range is very limited, and the currently known hosts include onion, leek, lisianthus, and alstroemeria (2). The virus is vectored by onion thrips (Thrips tabaci). Onion (Allium cepa) is grown as a winter crop in Georgia from September to April and is the only known host commercially grown in the region. However, the virus has been found across the onion-growing region in the state every year since its first occurrence during 2003 (3). Consequently, the virus must oversummer in other host(s) or its insect vector. Accordingly, samples of weeds were collected in the vicinity of onion fields and cull piles in the Vidalia region and tested for the presence of IYSV by a double-antibody sandwich (DAS)-ELISA (Agdia, Inc., Elkhart, IN). One of three nonsymptomatic spiny sowthistle samples tested positive by ELISA for IYSV. Total RNA was extracted from the leaf using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. Two microliters were used for reverse transcription (RT)-PCR with the forward primer (5'-TCAGAAATCGAGAAACTT-3') and reverse primer (5'-TAATTATATCTATCTTTCTTGG-3') for the IYSV nucleocapsid gene (1). A band of the expected size (approximately 800 bp) was obtained and sequenced. The sequence from the sowthistle (GenBank Accession No. EU078327) matched IYSV sequences from Georgia and Peru in a BLAST search in GenBank (closest matches with Accession Nos. DQ838584, DQ838592, DQ838593, and DQ658242). This is to our knowledge, the first confirmed report of IYSV infecting spiny sowthistle. The distribution of IYSV in sowthistle and its role as an oversummering host for IYSV is currently an on-going study. References: (1) L. du Toit et al. Plant Dis. 88:222, 2004. (2) D. H. Gent et al. Plant Dis. 90:1468, 2006. (3) S. W. Mullis et al. Plant Dis. 88:1285, 2004.

Response of Peanut, Pepper, Tobacco, and Tomato Cultivars to Two Biologically Distinct Isolates of Tomato spotted wilt virus.

Spotted wilt disease, caused by Tomato spotted wilt virus (TSWV), is an economically important disease in peanut, pepper, tobacco, and tomato in the southeastern United States. However, very little is known about the biological variability existent in the virus population. Fourteen isolates of TSWV collected in Georgia were evaluated for symptom severity. The majority of the isolates produced severe systemic necrosis. One mild (GATb-1) and one severe (GAL) isolate were further examined because of the distinct differences in their virulence and symptomatology on tobacco. GATb-1 caused a few chlorotic spots and mild systemic symptoms, whereas GAL produced a large number of local lesions and severe systemic necrosis. Distinct differences in the response of selected commercial cultivars of peanut, tobacco, and tomato to GATb-1 and GAL infection were observed. GAL was lethal to a widely grown tobacco cultivar, K326. Georgia Green, a field resistant peanut cultivar, and C11-2-39, a breeding line with the highest level of known resistance to TSWV, were more susceptible to GAL than to GATb-1. BHN 444, a newly released TSWV-resistant tomato cultivar, showed a resistant reaction, whereas Stiletto, a newly released TSWV-resistant pepper cultivar, was susceptible to both GATb-1 and GAL isolates. Information on the biological diversity of TSWV may be useful in developing more durable TSWV-resistant crops.

First Report of Pinaceae in Georgia Naturally Infected with Tomato spotted wilt virus.

In October 2004, three pine tree seedlings included in an ongoing survey of annual weeds elicited positive reactions for Tomato spotted wilt virus (TSWV [family Bunyaviridae, genus Tospovirus]) using double assay sandwich-enzyme linked immunosorbent assay (DAS-ELISA) (Agdia Inc. Elkhart, IN). All the seedlings appeared healthy with no visible adverse effects from the virus. Over the next 12 months, an additional 1,326 samples of various pine species representing different growth stages were screened for TSWV. Samples were comprised of local populations of Pinus elliottii Engelm., P. taeda L., and P. palustris P. Mill., with the majority (n = 886) of samples being seedlings collected from southern Georgia. Along with the seedlings, needles, stem sections, and roots from saplings, as well as needles from mature trees, were screened for the virus. Of the trees sampled, 5.35% (n = 71) tested positive for TSWV, and of the seedlings 6.77% (n = 60) tested positive. The DAS-ELISA positive threshold was obtained using a figure of three times the average plus two standard deviations of healthy negative pine tissue control absorbance readings at 405 nm. A number of saplings testing positive (n = 6) were marked for further evaluation, and the needles from these saplings consistently screened positive for TSWV in subsequent testing. Furthermore, several samples were processed in modified burlese funnels to detect the possible presence of thrips. No thrips were ever identified in any of the burlese funnel collections. Different tissue types (needles, roots, stem sections, and reproductive organs) were screened, but the virus was only detected in needles. This suggests that local infections are only at feeding sites of viruliferous thrips. The known thrips vectors for TSWV are not considered to be pine feeders, and there is no indication that pine trees are a reproductive reservoir for any local thrips species. However, pine-feeding thrips may also feed on known weed hosts, thus pines could be a perennial reservoir. Mechanical inoculations from surface-sterilized infected pine needles onto known TSWV indicator plants (Nicotiana glutinosa L., N. benthamiana, and Emilia sonchifolia L. (DC)) were inconsistent. Successful transmission occurred 24% of the time. To further verify serological data, total RNA extracts of pine sap were purified and subjected to immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) using primers specific to the nucleocapsid gene of TSWV (1). IC-RT-PCR was used due to the inability to obtain useful total RNA from the pine tissues. This may be due to a secondary metabolite interfering with the total RNA extraction protocol. The IC-RT-PCR products were analyzed with electrophoresis using 0.01% ethidium bromide stain in a 0.8% agarose gel. Amplicons produced at the expected size (bp = 774) were considered positive for TSWV. Several were sequenced and were consistent with known, local TSWV isolates. There is no indication that TSWV is detrimental to pine trees, but considering the widespread distribution of the genus Pinus and the potential of serving as a reservoir of TSWV, it may play a role in the overall epidemiology of TSWV in southern Georgia. Reference: (1) R. K. Jain et al. Plant Dis. 82:900, 1998.

First Report of Onion (Allium cepa) Naturally Infected with Iris yellow spot virus in Peru.

Onions have become an important export crop for Peru during the last few years. The onions produced for export are primarily short-day onions and include Grano- or Granex-type sweet onions. The first of two growing seasons for onion in Peru occurs from February/March until September/October and the second occurs from September/October to December/January. Iris yellow spot virus (IYSV [family Bunyaviridae, genus Tospovirus]), primarily transmitted by onion thrips (Thrips tabaci), has been reported in many countries during recent years, including the United States (1,2). In South America, the virus was reported in Brazil during 1999 (3) and most recently in Chile during 2005 (4). During 2003, an investigation of necrotic lesions and dieback in onions grown near the towns of Supe and Ica, Peru led to the discovery of IYSV in this region. Of 25 samples of symptomatic plants collected from five different fields near Supe, 19 tested strongly positive and an additional three tested weakly positive for IYSV using double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) (Agdia Inc., Elkhart, IN). None of the samples tested positive for Tomato spotted wilt virus (TSWV). A number of onions with necrosis and dieback symptoms were also observed during 2004 and 2005. During September 2005, 25 plants with symptoms suspected to be caused by IYSV or TSWV in the Supe and Casma valleys were collected and screened for both viruses using DAS-ELISA. All plants screened were positive for IYSV. There was no serological indication of TSWV infection in these samples. The positive samples were blotted onto FTA cards (Whatman Inc., U.K.) to bind the viral RNA for preservation and processed according to the manufacturer's protocols. The presence of IYSV was verified by reverse transcription-polymerase chain reaction (RTPCR) using (5'-TCAGAAATCGAGAAACTT-3') and (5'-TAATTATATCTATCTTTCTTGG-3') as forward and reverse primers (1), respectively. The primers amplify the nucleocapsid (N) gene of IYSV, and the RT-PCR products from this reaction were analyzed with gel electrophoresis with an ethidium bromide stain in 0.8% agarose to verify the presence of this amplicon in the samples. Subsequent to the September 2005 sampling, 72 additional samples from regions in northern and southern Peru were analyzed in the same manner. The amplicons obtained were cloned, sequenced, and compared with known IYSV isolates for further verification. Onions have become a significant export crop for Peru, and more research is needed to determine the impact of IYSV on the Peruvian onion export crop. To our knowledge, this is the first report of IYSV in onion in Peru. References: (1) L. du Toit et al. Plant Dis. 88:222, 2004. (2) S. W. Mullis et al. Plant Dis. 88:1285, 2004. (3) L. Pozzer et al. Plant Dis. 83:345, 1999. (4) M. Rosales et al. Plant Dis. 89:1245, 2005.

First Report of Tomato spotted wilt virus in Soybean (Glycine max) in Georgia.

Tomato spotted wilt virus (TSWV) is a member of the family Bunyaviridae and has a wide host range including important crops such as tomato, pepper, tobacco, peanut, and onion. In areas of Georgia, soybean (Glycine max) is double cropped between two onion crops and as a rotation crop with peanuts. Soybeans do not show any TSWV symptoms, and therefore, have not been tested on a large scale for the virus. However, because symptomless weed and crop plants provide a reservoir for TSWV and the thrips vectors (2), a survey was conducted during the summer of 2005 to evaluate the occurrence of TSWV in soybean. The survey took place in seven counties in southern Georgia with field sizes ranging between 0.4 and 20 ha (1 and 50 acres). Soybean cultivars included Haskell, DP7220, DP6770, Pioneer 97B52, and Vigoro V622NRR. Of 848 randomly selected plants tested using the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Agdia, Inc., Elkhart, IN), 6.6% tested positive for TSWV. Plants testing positive ranged from seedling to the pod-setting stages. Leaves and roots of several plants tested positive, indicating a systemic infection. Soybean plants testing positive using ELISA were blotted onto FTA cards (Whatman Inc., Brentford, UK) to bind viral RNA for preservation, and the blotted samples were processed according to the manufacturer's protocol. Reverse transcription-polymerase chain reaction using punch-outs from the FTA cards and TSWV nucleocapsid gene specific forward and reverse primers (5'-TTAAGCAAGTTCTGTGAG-3' and 5'-ATGTCTAAGGTTAAGCTC-3'), respectively (4), confirmed the identity of TSWV. TSWV has been found in soybean in other parts of the world (1) but has only been reported in the United States in a survey from Tennessee (3). To our knowledge, this is the first report of the occurrence of TSWV in soybean in Georgia. The role soybean plays as a reservoir or green bridge for thrips and TSWV is currently unknown. References: (1) A. R. Golnaraghi et al. Plant Dis. 88:1069, 2004. (2) R. L. Groves et al. Phytopathology 91:891, 2001. (3) B. S. Kennedy and B. B. Reddick. Soybean Genet. Newsl. 22:197, 1995. (4) H. R. Pappu et al. Tob. Sci. 40:74, 1996.

First Report of Tomato spotted wilt virus in Leek (Allium porrum) in the United States.

Tomato spotted wilt virus (TSWV) is a member of the family Bunyaviridae. It has many important crop hosts including tomato, pepper, tobacco, peanut, and onion. In Georgia, Vidalia onions (Allium cepa), a close relative of leek, can be infected by TSWV and Iris yellow spot virus (IYSV), which is another thrips-vectored tospovirus (2). For this reason, samples of leek transplants with virus-like symptoms in one field at the border of Georgia and Florida were tested for the presence of TSWV and IYSV. The transplants had been grown from seed in a greenhouse at the same location. The sampled plants exhibited extended bleaching of leaf tips and necrotic lesions. These symptoms were also seen on onion plants infected with TSWV and IYSV. The only natural infections of leek with IYSV have been reported thus far only from Reunion Island (4) and Slovenia (1), but to our knowledge, TSWV has not been reported as a pathogen of leek. Green tissue near the necrotic lesions and bleached tips of one symptomatic leaf per plant was sampled and analyzed using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Agdia, Inc., Elkhart, IN). Of 90 plants tested, eight were positive for TSWV and none were positive for IYSV. Leek samples testing positive using ELISA were blotted onto FTA cards (Whatman Inc., Brentford, UK) to bind viral RNA for preservation and then processed according to the manufacturer's protocol. Punch-outs from the FTA cards were used for reverse transcription polymerase chain reaction (RT-PCR) with the TSWV-specific forward primer (5'-TTAAGCAAGTTCTGTGAG-3') and reverse primer (5'-ATGTCTAAGGTTAAGCTC-3') (3) to confirm the identity of TSWV. The primers are specific to the viral nucleocapsid gene. An amplicon of the expected size (774 bp) was produced from TSWV ELISA-positive leek plants, but not from healthy controls. TSWV has been found in many plants worldwide, but to our knowledge this is the first report of TSWV infecting leek. The effect that TSWV has on leek production is currently unknown. References: (1) D. A. Benson et al. Nucleic Acids Res. 1:32 (Database issue):D23-6, 2004. (2) S. W. Mullis et al. Plant Dis. 88:1285, 2004. (3) H. R. Pappu et al. Tob. Sci. 40:74, 1996. (4) I. Robène-Soustrade et al. Online publication. New Dis. Rep. 11, 2005.

Root-knot nematode management in double-cropped plasticulture vegetables.

Combination treatments of chisel-injected fumigants (methyl bromide, 1,3-D, metam sodium, and chloropicrin) on a first crop, followed by drip-applied fumigants (metam sodium and 1,3-D +/- chloropicrin) on a second crop, with and without oxamyl drip applications were evaluated for control of Meloidogyne incognita in three different tests (2002 to 2004) in Tifton, GA. First crops were eggplant or tomato, and second crops were cantaloupe, squash, or jalapeno pepper. Double-cropped vegetables suffered much greater root-knot nematode (RKN) pressure than first crops, and almost-total yield loss occurred when second crops received no nematicide treatment. On a first crop of eggplant, all fumigants provided good nematode control and average yield increases of 10% to 15 %. On second crops, higher application rates and fumigant combinations (metam sodium and 1,3-D +/- chloropicrin) improved RKN control and increased yields on average by 20% to 35 % compared to the nonfumigated control. Oxamyl increased yields of the first crop in 2003 on average by 10% to 15% but had no effect in 2004 when RKN failed to establish itself. On double-cropped squash in 2003, oxamyl following fumigation provided significant additional reduction in nematode infection and increased squash yields on average by 30% to 75%.

Relationship of Isolate Origin to Pathogenicity of Race 0 and 1 of Phytophthora parasitica var. nicotianae on Tobacco Cultivars.

Flue-cured tobacco cultivars were evaluated for their reaction to race 0 and race 1 of Phytophthora parasitica var. nicotianae, the incitant of the disease tobacco black shank. Seventeen commercial tobacco cultivars having resistance derived from Fla 301 or a combination of Fla 301 and Fla 105 were subjected to root or stem inoculation by 22 different isolates of P. parasitic var. nicotianae collected from across the Georgia tobacco-growing belt. An adapted stem inoculation technique using field-grown tobacco indicator cvs. K-326, NC-71, Coker 371 Gold, and the breeding line NC-1071 was used to determine races of P. parasitica var. nicotianae. Typically, in greenhouse evaluations, cultivars stem inoculated with race 1 of P. parasitica var. nicotianae were killed. Cultivars that had resistance from both Fla 301 and Fla 105 (Ph gene) were not killed, with few exceptions, when inoculated with race 0. Fifty-seven tobacco specimens having black shank symptoms from the Georgia and Florida tobacco belt were evaluated for P. parasitica var. nicotianae race using the adapted stem inoculation technique. Of the samples evaluated from commercial tobacco fields, 83% yielded race 1 compared with a similar evaluation made in 1994, where only 16% of the samples yielded race 1. The increase in race 1 incidence may be related to the increase in use of cultivars which have the Ph gene for resistance. The use of rotations and metalaxyl or mefenoxam may be required where race 1 is found.

First Report of Yellow Nutsedge (Cyperus esculentus) and Purple Nutsedge (C. rotundus) in Georgia Naturally Infected with Impatiens necrotic spot virus.

Impatiens necrotic spot virus (INSV), family Bunyaviridae, genus Tospovirus, is an emerging virus found mostly in ornamentals under greenhouse production. INSV has been detected in peanut (Arachis hypogaea L.) in Georgia and Texas (3) and recently in tobacco (Nicotiana tabacum L.) in the southeastern United States (2) but little is known about INSV distribution and impact on these crops. Noncrop plant hosts are likely to contribute to disease spread by serving as reservoirs for the virus and reproductive hosts for thrips (Frankliniella occidentalis Pergande), which transmit the virus. Yellow nutsedge, a native of North America, and purple nutsedge introduced from Eurasia, are considered serious weed problems in the southeastern United States. To date, there are no reports of natural INSV infections in these weeds. A survey was conducted at two research farms in Tift County, Georgia to determine if yellow and purple nutsedge plants were naturally infected with Tomato spotted wilt virus (TSWV) and INSV. The first field at the Black Shank Farm had been planted with flue-cured tobacco K-326 earlier in the year and fallow at the time of sampling. The second field at the Ponder Farm was planted at the time of sampling with yellow squash (Cucurbita pepo L.) and cabbage (Brassica oleracea L.). In early October 2002, 90 nutsedge plants were taken at random from each site. Leaf and root tissues of each of the nutsedge plants were tested for TSWV and INSV using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) alkaline phosphatase antisera kits (Agdia Inc., Elkhart, IN). No visible symptoms of INSV or TSWV were observed. Samples from the field at the Black Shank Farm resulted in 2 of 26 positive for INSV in purple nutsedge plants and 6 of 64 in yellow nutsedge plants. At the Ponder Farm, 3 of 12 were positive for INSV in purple nutsedge plants and 14 of 78 in yellow nutsedge plants. None of the samples in either site tested positive for TSWV. The DAS-ELISA positive samples were verified for INSV using reverse transcription-polymerase chain reaction (RT-PCR) as previously described by Dewey et al. (1). Total RNA extracts were obtained from the DAS-ELISA positive nutsedge samples using RNeasy extraction kits (Qiagen Inc., Valencia, CA). The RT-PCR was carried out with primer 1F: 5'-TCAAG(C/T) CTTC(G/T)GAA(A/G)GTGAT 3' (1) and primer 2R: 5'-ATGAACAAAGCAAAGATTACC 3' specific to the 3' end of the INSV N gene open reading frame (GenBank Accession No. NC003624). DAS-ELISA negative tissues of Cyperus esculentus L. and Emilia sonchifolia (L.) DC and an E. sonchifolia DAS-ELISA positive for INSV were included in the reactions as controls. All of the DAS-ELISA positive nutsedge samples yielded an amplification product with the expected size of 298 bp when PCR products were resolved by agarose (0.7%) gel electrophoresis. The relatively high occurrence of INSV found in the sampled fields may explain the recent increase in incidence of INSV in susceptible field crops. Although yellow nutsedge is more common than purple nutsedge in North America, the potential for dispersal of INSV in both species could be significant because of the nature of nutsedge tuber survival and spreading capabilities. References: (1) R. A. Dewey et al. J. Virol. Methods 56:19, 1996. (2) N. Martínez-Ochoa et al. On-line publication. doi:10.1094/PHP-2003-0417-01-HN. Plant Health Progress, 2003. (3) S. S. Pappu et al. Plant Dis. 83:966,1999.

Management of Tomato spotted wilt virus in Flue-Cured Tobacco with Acibenzolar-S-Methyl and Imidacloprid.

Tomato spotted wilt virus (TSWV) continues to be a major production constraint in flue-cured tobacco in Georgia. Pretransplant applications of acibenzolar-S-methyl (Actigard) and imidacloprid (Admire and Provado) were evaluated in field trials, singly and in combination, in four locations in 1999. Acibenzolar-S-methyl was also evaluated for its effect on growth and yield, potential phytotoxicity, and activity against tobacco blackshank incited by Phytophthora parasitica var. nicotianae. Acibenzolar-S-methyl alone and with imidacloprid significantly reduced TSWV incidence in all four locations, but the disease incidence in the imidacloprid-treated plots was lower in only one of the four sites. Applications of acibenzolar-S-methyl starting at 4 weeks posttransplant had no effect on TSWV incidence, size, or yield of tobacco compared with nontreated plots. Acibenzolar-S-methyl did not reduce blackshank in a field trial. In the greenhouse, rates of acibenzolar-S-methyl ranging from 0.25 to 8 g a.i. per 7,000 plants showed reductions in growth that did not seem to be related to rate. Pretransplant applications of acibenzolar-S-methyl were critical to the suppression of TSWV, while initial applications made posttransplant had no effect, suggesting that plants must be protected prior to introduction into the field.

Nucleocapsid Gene-Mediated Transgenic Resistance Provides Protection Against Tomato spotted wilt virus Epidemics in the Field.

ABSTRACT Transformation of plants with the nucleocapsid (N) gene of Tomato spotted wilt tospovirus (TSWV) provides resistance to disease development; however, information is lacking on the response of plants to natural inoculum in the field. Three tobacco cultivars were transformed with the N gene of a dahlia isolate of TSWV (TSWV-D), and plants were evaluated over several generations in the greenhouse. The resistant phenotype was more frequently observed in 'Burley 21' than in 'KY-14' or 'K-326', but highly resistant 'Burley 21' transgenic lines were resistant to only 44% of the heterologous TSWV isolates tested. Advanced generation (R(3) and R(4)) transgenic resistant lines of 'Burley 21' and a 'K-326' F(1) hybrid containing the N genes of two TSWV isolates were evaluated in the field near Tifton, GA, where TSWV is endemic. Disease development was monitored by symptom expression and enzyme-linked immunosorbent assay (ELISA) analysis. Whereas incidence of TSWV infection in 'Burley 21' susceptible controls was 20% in 1996 and 62% in 1997, the mean incidence in transgenic lines was reduced to 4 and 31%, respectively. Three transgenic 'Burley 21' lines were identified that had significantly lower incidence of disease than susceptible controls over the two years of the study. In addition, the rate of disease increase at the onset of the 1997 epidemic was reduced for all the 'Burley 21' transgenic lines compared with the susceptible controls. The 'K-326' F(1) hybrid was as susceptible as the 'K-326' nontransformed control. ELISA analysis demonstrated that symptomless plants from the most resistant 'Burley 21' transgenic lines accumulated detectable nucleocapsid protein, whereas symptomless plants from more susceptible lines did not. We conclude that transgenic resistance to TSWV is effective in reducing incidence of the disease in the field, and that accumulation of transgene protein may be important in broad-spectrum resistance.

Stem and Root Resistance to Tobacco Black Shank.

Stem lesion development in the absence of root decay in tobacco black shank caused by Phytophthora parasitica var. nicotianae has become common in the Coastal Plain tobacco growing area in Georgia. All aboveground symptoms of wilting, blackening of lower stem, and destruction of the pith can occur on tobacco without or with minor root decay. This type of black shank disease development occurred in 14 of 15 locations evaluated and accounted for about 30% of diseased plants. Cultivars with Florida 301-derived resistance had very low stem resistance to race 0 of the pathogen. However, root inoculations of these cultivars resulted in disease reactions typical of those expected in vivo in Georgia. Cultivars Coker 371-Gold and NC 71 and the breeding line 1071 demonstrated high resistance to inoculation with race 0 of P. parasitica var. nicotianae in both the stem and the roots, but they were susceptible when stem-inoculated with race 1 of the pathogen. Severity of root decay was isolate dependent.

Chemigation for Control of Black Shank-Root-knot Complex and Weeds in Tobacco.

Tank mixes of a fungicide (metalaxyl) and a nematicide (fenamiphos) with herbicides (isopropalin or pendimethalin) and an insecticide (chlorpyrifos) were applied by soil incorporation or irrigation to control the black shank-root knot complex and weeds on four tobacco cultivars. The disease complex was more severe on cultivars McNair 944, NC-2326, and K-326 than on Speight G-70. The disease complex was reduced (P

Inhibition of growth ofPhytophthora parasitica var.nicotianae by aromatic acids and coumarins in a laboratory bioassay.

Hydroxy-, dihydroxy-, trihydroxy-, methoxy-, dimethoxy-, hydroxy-+methoxy-, amino-, chloro-, and nitro-substituted benzoic, phenylacetic, phenylpropanoic, and phenylpropenoic (cinnamic) acids were evaluated for activity against the growth ofPhytophthora parasitica var.Nicotianae, Races 0 and 1, in a laboratory bioassay. Several substituted coumarins were also tested. In general, for Race 0, the phenylpropenoic acids were more active (on a millimolar basis), than the corresponding benzoic, phenylacetic, or phenylpropionic acids (9 of 14 series). Among the most active acids wereo-hydroxycinnamic and the chloro- and methoxycinnamic acids. The activities of unsubstituted benzoic and phenylpropionic acids were comparable to the most active compounds tested. Monohydroxyaromatic acids were more active than most dihydroxy acids of the same chain length. Dihydro-3,4-dihydroxycinnamic acid was slightly more active than the corresponding cinnamic acid, while the reverse was true for the mono-p-hydroxycinnamic acid versusp-hydroxyphenylpropionic acid. Coumarin was more active than its hydroxy, methyl, hydroxymethyl, or methoxy derivatives. In general, Race 1 was even more significantly affected by the aromatic acids. Glycosylated coumarins were inactive in the bioassay, compared to their aglycones.