Field Evaluations of Leaf Spot Resistance and Yield in Peanut Genotypes in the United States and Bolivia.

Field experiments were conducted in 2002 to 2006 to characterize yield potential and disease resistance in the Bolivian landrace peanut (Arachis hypogaea) cv. Bayo Grande, and breeding lines developed from crosses of Bayo Grande and U.S. cv. Florida MDR-98. Diseases of interest included early leaf spot, caused by the fungus Cercospora arachidicola, and late leaf spot, caused by the fungus Cercosporidium personatum. Bayo Grande, MDR-98, and three breeding lines, along with U.S. cvs. C-99R and Georgia Green, were included in split-plot field experiments in six locations across the United States and Bolivia. Whole-plot treatments consisted of two tebuconazole applications and a nontreated control. Genotypes were the subplot treatments. Area under the disease progress curve (AUDPC) for percent defoliation due to leaf spot was lower for Bayo Grande and all breeding lines than for Georgia Green at all U.S. locations across years. AUDPC for disease incidence from one U.S. location indicated similar results. Severity of leaf spot epidemics and relative effects of the genotypes were less consistent in the Bolivian experiments. In Bolivia, there were no indications of greater levels of disease resistance in any of the breeding lines than in Bayo Grande. In the United States, yields of Bayo Grande and the breeding lines were greater than those of the other genotypes in 1 of 2 years. In Bolivia, low disease intensity resulted in the highest yields in Georgia Green, while high disease intensity resulted in comparable yields among the breeding lines, MDR-98, and C-99R. Leaf spot suppression by tebuconazole was greater in Bolivia than in the United States. This result indicates a possible higher level of fungicide resistance in the U.S. population of leaf spot pathogens. Overall, data from this study suggest that Bayo Grande and the breeding lines may be desirable germplasm for U.S. and Bolivian breeding programs or production.

Epidemiology and management of tomato spotted wilt in peanut.

Tomato spotted wilt caused by thrips-vectored tomato spotted wilt virus (TSWV) is a very serious problem in peanut (Arachis hypogaea L.) production. TSWV and the thrips Frankliniella fusca and Frankliniella occidentalis, which vector the virus, present a difficult and complicated challenge from the perspectives of both epidemiology and disease management. Simply controlling the vector typically has not resulted in control of spotted wilt. No single measure can currently provide adequate control of spotted wilt where severe epidemics occur. However, interdisciplinary investigations have resulted in development of integrated management systems that make use of moderately resistant cultivars and chemical and cultural practices, each of which helps to suppress spotted wilt epidemics. Such systems have been successfully deployed in many areas for minimizing losses to this disease. The development of a spotted wilt risk index has aided greatly in relaying information on the importance of using an integrated approach for managing this disease.

Differential Response of Selected Peanut (Arachis hypogaea) Genotypes to Mechanical Inoculation by Tomato spotted wilt virus.

Screening of peanut germ plasm for resistance to Tomato spotted wilt virus (TSWV) has been largely inefficient due to the lack of a screening technique based on mechanical transmission of the virus under controlled environmental conditions. We have studied the reaction of three peanut cultivars (Georgia Green, Georgia Runner, C-99R) and one breeding line (C11-2-39) using a highly efficient mechanical inoculation procedure. The disease response was studied at two temperature regimes, 25 to 30°C (low temperature) and 30 to 37°C (high temperature). Based on percent transmission, symptomatology, distribution of TSWV, and relative levels of TSWV nucleocapsid (N) protein, Georgia Runner and Georgia Green were found to be susceptible, whereas C-99R and C11-2-39 were resistant. Of the four genotypes tested, C11-2-39 had the highest level of resistance to TSWV. The results correlated with the field performance of the genotypes except in the case of Georgia Green, which could not be distinguished from TSWV-susceptible Georgia Runner. Exposure of the inoculated plants to higher temperature (30 to 37°C) resulted in a better resistant response as reflected by reduced systemic infection, localized symptom expression, restricted viral movement, and reduced levels of TSWV antigen. To our knowledge, this is the first report of differential response of peanut genotypes to TSWV using mechanical inoculation. The four peanut genotypes should be useful as reference standards for the initial screening and identification of sources of TSWV resistance in peanut germ plasm.

First Report of Natural Infection of Peanut (Groundnut) by Impatiens Necrotic Spot Tospovirus (Family Bunyaviridae).

Impatiens necrotic spot tospovirus (INSV) of the family Bunyaviridae is an important viral pathogen of ornamentals and a major constraint in the greenhouse industry (2). Evidence of natural infection of peanut (groundnut, Arachis hypogaea L.) by INSV was found in samples collected from three sites in Frio County, TX, and one site each in Mitchell and Tift counties, GA, during October 1998. Roots from several plants were tested by enzyme-linked immunosorbent assay for tomato spotted wilt tospovirus (TSWV) and INSV. Symptoms on individual mature plants positive for INSV were the same as those associated with late-season TSWV infections: plants appeared yellow and wilted, internal taproot and crown were necrotic, and plant death resulted (1). At one Texas site, three of five composite samples were positive only for INSV. One composite sample at a second Texas site was positive for both TSWV and INSV. Double infections were found in three of four TSWV-positive samples at a third Texas site. In Mitchell County, GA, three of four samples tested were positive only for TSWV, and one was positive for both TSWV and INSV. In Tift County, GA, 11 of 23 samples tested were positive only for INSV, whereas 4 were positive only for TSWV. Double infections were found in 5 of 23 samples. The presence of INSV in the sample from Mitchell County was verified by immunocapture-polymerase chain reaction (PCR) (4). The apparently low titer of INSV in the doubly infected plant necessitated two cycles of PCR for detection of INSV sequences. A primer pair that can amplify most tospoviruses was used for the first PCR cycle (3). Using the PCR product obtained, a second PCR cycle was performed with one tospovirus-specific and one INSV-specific primer. This approach resulted in a product of the expected size (≈298 bp). The PCR product was cloned in a pGEM-T vector and sequenced. Comparisons indicated the sequence obtained from the infected peanut sample from Georgia was 99% identical to the respective S RNA region from known INSV isolates. Serological and molecular sequence data suggest the peanut samples were infected by INSV. Future surveys and screenings of peanut plants for spotted wilt disease should include a test for INSV. References: (1) A. K. Culbreath et al. Plant Dis. 75:863, 1991. (2) M. Daughtrey et al. Plant Dis. 81:1220, 1997. (3) R. Dewey et al. Virus Genes 13:255, 1996. (4). R. K. Jain et al. Plant Dis. 82:900, 1998.

First Report on the Multiplication of Tomato Spotted Wilt Tospovirus in Tobacco Thrips, Frankliniella fusca.

In Georgia, tomato spotted wilt tospovirus (TSWV) causes significant losses in peanut, tobacco, tomato, and pepper. Transmission of TSWV in Georgia primarily is by tobacco thrips (TT), Frankliniella fusca (Hinds), and western flower thrips, F. occidentalis (Pergande), with TT being the predominant vector species in peanut (2). TSWV must be acquired at the larval stage for the adult to transmit the virus. Detection of NSs (a non-structural TSWV protein present only following virus replication) in thrips by enzyme-linked immunosorbent assay (ELISA) is a reliable indicator that the virus had multiplied in the vector and thus the vector is competent to transmit TSWV. While this has been accomplished with F. occidentalis (1), information is lacking for F. fusca, the predominant vector in Georgia and other states in the Southeast. Thus, the nature of the TSWV-TT association was investigated and the proportion of transmitters in a field population determined in 1,436 individual adult TT collected from sticky cards positioned in selected peanut fields in south Georgia. Additionally, 650 larvae collected from volunteer peanut plants were reared to adults in the laboratory and the resulting 295 adult TT were individually evaluated by ELISA. Of those collected from the sticky cards, NSs was detected in 8% of the adult insects, indicating that the virus had multiplied in TT. NSs was not detected in control TT that had no access to the virus. Of the adult TT that emerged from larvae collected from volunteer peanuts, 6.1% were positive for NSs. Our study provides the first immunological evidence that TSWV multiplies in TT. References: (1) M. D. Bandla et al. Phytopathology 84:1427, 1994. (2) J. R. Chamberlin et al. J. Econ. Entomol. 86:40, 1993.

Differentiation of Biologically Distinct Peanut Stripe Potyvirus Strains by a Nucleotide Polymorphism-Based Assay.

A necrotic strain of peanut stripe potyvirus (PStV-Ts) was used to design and test strain-differentiating oligonucleotides. The 3' region of PStV-Ts, including a part of the NIb region, the complete coat protein (CP) gene, and the 3'-untranslated region, was cloned and sequenced. PStV-Ts had a high degree of sequence identity (92 to 95%) to the known non-necrotic (blotch) strains both at the nucleotide and amino acid sequence levels. Nucleotide sequence differences unique to the necrotic strain were identified when compared to the available non-necrotic isolates of PStV. Nucleotide polymorphism in the CP gene sequences was utilized in designing oligonucleotides that were specific to the necrotic strain, and were employed in an assay to differentiate the necrotic strain from non-necrotic. The 3' end mismatch in the oligonucleotides contributed in particular to the differentiation of the strains. This approach facilitated rapid, sensitive, and reliable detection and differentiation of PStV strains.

Molecular Diagnosis of Tomato Spotted Wilt Tospovirus Infection of Peanut and Other Field and Greenhouse Crops.

Tomato spotted wilt tospovirus (TSWV) is a major disease constraint to peanut, tomato, pepper, and tobacco production in Georgia. Rapid molecular diagnosis of TSWV infection in peanut and its molecular studies were severely hampered by the lack of practical and rapid procedures for the extraction and amplification of the genomic nucleic acid. To circumvent this technical constraint, we adapted an immunocapture-procedure (ICP) for enriching the peanut tissue extracts for TSWV, and combined the ICP with a single-buffer, one-tube reverse transcription polymerase chain reaction (RT-PCR) to achieve rapid and reliable amplification of TSWV sequences from peanut. Both leaf and root tissue of peanut provided PCR-quality templates. Immunocapture, RT, and PCR were done in the same tube, allowing higher throughput. The technique was applicable to a wide range of TSWV-susceptible crops such as tomato, pepper, tobacco, gloxinia, and impatiens. Primers derived from the nucleocapsid protein gene as well as from the large RNA of the viral genome were able to amplify the target sequences in a highly specific and reproducible manner. This approach facilitated rapid molecular typing of natural populations of TSWV in Georgia.

Peanut-cotton-rye rotations and soil chemical treatment for managing nematodes and thrips.

In the southeastern United States, a cotton-peanut rotation is attractive because of the high value and extensive planting of both crops in the region. The objective of this experiment was to determine the effects of cotton-peanut rotations, rye, and soil chemical treatments on management of plant-parasitic nematodes, thrips, and soilborne fungal diseases and on crop yield. Peanut-cotton-rye rotations were conducted from 1988 to 1994 on Tifton loamy sand (Plinthic Kandiudult) infested primarily with Meloidogyne incognita race 3, Belonolaimus longicaudatus, Sclerotium rolfsii, Rhizoctonia solani, and Fusarium oxysporum. Continuous peanut, continuous cotton, cotton-peanut rotation, or peanut-cotton rotation were used as main plots; winter rye or fallow as sub-plots; and cotton with and without aldicarb (3.36 kg a.i./ha), or peanut with and without aldicarb (3.36 kg a.i./ha) plus flutolanil (1.12 kg a.i./ha), as sub-sub-plots. Population densities of M. incognita and B. longicaudatus declined rapidly after the first crop in continuous peanut and remained low thereafter. Neither rye nor soil chemical treatment affected M. incognita or B. longicaudatus population density on peanut or cotton. Cotton and peanut yields from the cotton-peanut rotation were 26% and 10% greater, respectively, than those from monoculmre over the 7-year study. Cotton and peanut yields were improved 9% and 4%, respectively, following rye vs. fallow. Soil chemical treatments increased yields of cotton 23% and peanut 32% over those of untreated plots. Our data demonstrate the sustainable benefits of using cotton-peanut rotations, winter rye, and soil chemical treatments to manage plant-parasitic nematodes and other pests and pathogens and improve yield of both cotton and peanut.

Field Response of New Peanut Cultivar UF 91108 to Tomato Spotted Wilt Virus.

Epidemics of spotted wilt, caused by tomato spotted wilt tospovirus (TSWV), were monitored in field plots of the new runner-type peanut (Arachis hypogaea) cv. UF 91108, in advanced breeding line F 84 × 9B-4-2-1-1-2-b2-B, in runner-type peanut cvs. Southern Runner and Florunner, and in Virginia-type cv. NC-V11 at two locations in 1994 and 1995. Epidemics of spotted wilt were suppressed in UF 91108 compared to the standard runner-type cv. Florunner. Final disease incidence, standardized area under the disease progress curve values, and final disease intensity ratings were lower in UF 91108 than in Florunner and were similar to those in the moderately resistant cv. Southern Runner. Results indicate that new cv. UF 91108 represents a new potential tool for management of spotted wilt in peanut production areas of the southeastern United States. UF 91108 is the first peanut cultivar in which an oil composition of approximately 65% oleic acid is combined with a moderate level of field resistance to TSWV. Epidemics of spotted wilt also were suppressed in breeding line F 84 × 9B-4-2-1-1-2-b2-B. Across the four tests, the effects of NC-V11 on epidemic development, final incidence, and spotted wilt intensity ratings were not consistent relative to the other genotypes. Use of final disease intensity ratings provided separation of the genotypes similar to use of final incidence of spotted wilt. Assessment values by these two methods were highly correlated. In three of four experiments, final disease intensity ratings were more closely correlated with pod yield than was final incidence. The new intensity rating method described in this paper requires much less time and effort than determining disease incidence and may be a practical alternative to individual plant assessment for characterization of genotype responses to TSWV.

Factors regulating baculovirus late and very late gene expression in transient-expression assays.

Eighteen genes of Autographa californica nuclear polyhedrosis virus are necessary and sufficient to transactivate expression from the late vp39 promoter in transient-expression assays in SF-21 cells. These 18 genes, known as late expression factor genes (lefs), are also required to transactivate the very late promoter of the polyhedrin gene, polh, but expression from this promoter is relatively weak compared with expression from the vp39 promoter. To further define the factors required for late and very late promoter expression, we first determined that the eighteen lefs were also required for expression from two other major baculovirus promoters: the late basic 6.9-kDa protein gene, p6.9, and the very late 10-kDa protein gene, p10. We next examined the effect of the very late expression factor 1 gene (vlf-1), a gene previously identified by analysis of a temperature-sensitive mutant, in the transient expression assay and found that vlf-1 specifically transactivated the two very late promoters but not the two late promoters. We then surveyed the Autographa californica nuclear polyhedrosis virus genome for additional genes which might specifically regulate very late gene expression; no additional vlf genes were detected, suggesting that VLF-1 is the primary regulator of very late gene expression. Finally, we found that the relative contribution of the antiapoptosis gene p35, which behaves as a lef in these transient-expression assays, depended on the nature of the other viral genes provided in the cotransfection mixtures, suggesting that other viral genes also contribute to the ability of the virus to block apoptosis.

Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression.

We report the identification of four additional genes of the Autographa californica nuclear polyhedrosis virus involved in expression from a late baculovirus promoter in transient expression assays. Three of these genes, p35, 39K, and p47, have been previously described. The role of the p35 gene product in late gene expression may be related to its ability to block apoptosis, since two other baculovirus genes also known to block apoptosis, Cp-iap and Op-iap, were able to functionally replace p35 in the transient expression assay. The requirement for p47 in this assay confirms its role in late gene expression, a role previously established by characterization of a temperature-sensitive mutant of p47, while the requirement for 39K may be related to its known association with the virogenic stroma. The fourth gene identified as a late expression factor gene, lef-11, was located immediately upstream of 39K and is predicted to encode a 13-kDa polypeptide. When plasmids containing these 4 genes were cotransfected with plasmids containing the 14 genes previously identified as late gene expression factors, the level of expression from the late capsid promoter was similar to that observed for a library of clones representing the entire viral genome. The genes provided by these 18 plasmids thus represent the viral genes necessary and sufficient to support expression from a late viral promoter in this transient expression assay.

A baculovirus gene involved in late gene expression predicts a large polypeptide with a conserved motif of RNA polymerases.

We have identified and sequenced a novel baculovirus gene, late expression factor eight gene (lef-8), of Autographa californica nuclear polyhedrosis virus that is necessary for efficient expression from late and very late virus gene promoters in a transient expression assay. The predicted gene product, LEF-8, has a molecular mass of 102 kDa and contains a conserved sequence motif, GXKX4HGQ/NKG, found in DNA-directed RNA polymerases throughout the animal, plant, and microbial kingdoms.

Identification of lef-7: a baculovirus gene affecting late gene expression.

We have examined a 15-kb region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome, from 66 to 78 map units, for the presence of genes which transactivate expression from late and very late viral promoters in transient expression assays. One gene in this region activated reporter gene expression approximately six- to eightfold when the reporter gene was under the control of the late promoter of the major capsid protein gene, vp39, or the very late promoter of the polyhedrin gene, polh, but not when the reporter gene was under the control of the early promoter of etl, a homolog of proliferating cell nuclear antigen. The sequence of the predicted polypeptide product of this gene, lef-7, shared no obvious sequence homology to other sequences in available databases. Transcriptional analysis indicated that lef-7 was transcribed early in infection from an initiation site 14 to 16 bp upstream of the putative translational start site and was also transcribed late in infection from an initiation site(s) further upstream. The lef-7 promoter and the promoter of another previously defined late expression factor, lef-3, were both dependent on the multifunctional transregulatory gene, ie-1, for activity in transient expression assays. While sequencing the region of the AcMNPV genome containing lef-7, we also found a 215-codon open reading frame (ORF-215) approximately 1 kb downstream of lef-7 with sequence homology to eIF2 alpha kinases (e.g., rabbit eIF2 alpha and yeast GCN-2 kinase). However, only the six C-terminal conserved domains of protein kinases were present in the predicted ORF-215 product and several of these domains varied from the consensus sequence. ORF-215 did not strongly influence expression from the vp39 promoter-controlled reporter gene in the transient expression assays employed.