A. K. Culbreath

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.

Development of a Method of Risk Assessment to Facilitate Integrated Management of Spotted Wilt of Peanut

Tomato spotted wilt virus (TSWV) is one of a growing list of tospoviruses adversely affecting agriculture worldwide (36,38). Spotted wilt, the disease caused by TSWV, was first observed in peanut (Arachis hypogaea L.) growing regions of the southeastern United States in 1986 (41), and its importance steadily increased in Georgia through 1997 to the extent that annual losses exceeded

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

40 million. Since 1997, annual losses have sharply declined to approximately

Factors Affecting Mechanical Transmission of Tomato spotted wilt virus to Peanut (Arachis hypogaea)

10 million in 2000 (60) and

Integrated Disease Management of Leaf Spot and Spotted Wilt of Peanut

3.3 million in 2003 (15). The only known means of virus transmission is via vectors belonging to a few species of thrips (36,50). Only first instar larvae of Frankliniella occidentalis (Pergande), one vector species, can acquire the virus from an infected plant (57). After acquisition, the virus replicates in the vector and the viruliferous thrips is capable of transmission for the duration of its life (50,56,59). In Georgia, the primary vectors are tobacco thrips, F. fusca (Hinds), and western flower thrips, F. occidentalis (53). Most spotted wilt in peanut is thought to be the result of primary transmission, but some secondary transmission probably occurs as well (22), mostly by F. fusca, which readily reproduces on peanut (53,54). Peanut plants may initiate symptom expression at any stage of plant development (6), but severe stunting (Fig. 1) typically occurs on younger plants, whereas the initiation of symptoms on older plants typically results in generalized yellowing and/or localized leaf mottling (Fig. 2). Linear regression indicates that both number of seed and seed yield per infected plant increase with time from planting until symptom expression (27). Incidence and severity of tomato spotted wilt of peanut have been extremely variable in Georgia peanut fields (11). Research programs conducted since 1990 have identified several contributing factors affecting disease severity and have provided estimates of their relative importance. Some factors are more important than others, but no single factor can be manipulated to control the disease (26). Using available research data, a spotted wilt risk index was developed as a means of helping peanut growers to assess risk levels associated with specific combinations of production practices and thereby to avoid high-risk situations. Pertinent production practices were assigned point values that were weighted relative to their influence on final spotted wilt severity. As new research data became available, the index was modified and validated with extensive small plot trials and on-farm surveys. This article describes how the index was developed and modified and reports the results of on-farm surveys used to validate the index for peanut grown under a wide array of production practices. Factors Affecting Severity of Tomato Spotted Wilt in Peanut Peanut cultivar. Soon after spotted wilt became established in Georgia, the cultivar ‘Southern Runner’ was noted to have about half the incidence of that seen on the more widely grown ‘Florunner’ (5,28). This serendipitous discovery was followed by the discovery of a series of other varieties, some of which were produced from crosses with ‘Southern Runner’, having similar levels of resistance. ‘Georgia Browne’ (25), ‘Georgia Green’ (29), ‘Florida MDR 98’ (32), ‘ViruGard’ (31), ‘C99R’ (30,40,58), and several advanced breeding lines (29,30) have been shown to exhibit lower incidence of spotted wilt than ‘Florunner’ and other equally susceptible cultivars. The mechanism of resistance exhibited by these cultivars is unknown, but since thrips populations on resistant cultivars do not appear to be significantly lower than those on susceptible cultivars, differences in cultivar susceptibility are not thought to be due to differential preference by vectors (25,28–31). Planting date. Prior to severe outbreaks of spotted wilt in Georgia, planting date was found to influence the incidence of the disease on peanuts grown in southern Texas (47), where peanuts planted early and late in the normal planting season tended to have more spotted wilt than peanuts planted in the middle of the planting season, and those planted within a recommended “window” expressed less severe symptoms. Although actual planting dates are slightly different, a similar trend was Corresponding author: Steve L. Brown E-mail: bugbrown@arches.uga.edu

Analysis of Gene Expression Profiles in Leaf Tissues of Cultivated Peanuts and Development of EST-SSR Markers and Gene Discovery

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.

Identification of QTLs associated with oil content and mapping FAD2 genes and their relative contribution to oil quality in peanut (Arachis hypogaea L.)

Evaluation of peanut germ plasm for Tomato spotted wilt virus (TSWV) resistance has been slowed by the difficulty in achieving a high rate of mechanical transmission of the virus to peanut. In this study, improvements were made and a highly efficient mechanical transmission protocol was developed. Several factors that affect the transmission efficiency were identified. Use of two antioxidants (sodium sulfite and mercaptoethanol) and two abrasives (Celite and Carborundum) and application of the inoculum by rubbing with a cotton swab dipped in the inoculum as well as pricking with an inoculation needle resulted in a significantly higher transmission rate. The most susceptible growth stage of peanut to TSWV inoculation was 2 to 3 days after germination (6 to 7 days after planting). The inoculation protocol consistently resulted in a higher percentage of infected plants from different sources of inoculum such as infected peanut, tobacco, and tomato.

Fungicide dissipation and impact on metolachlor aerobic soil degradation and soil microbial dynamics.

Field experiments were carried out to evaluate the effects of integrated management of early leaf spot, caused by Cercospora arachidicola, and spotted wilt, caused by Tomato spotted wilt virus (TSWV), on peanut (Arachis hypogaea) using host resistance, two tillage systems, and varying fungicide programs. Effects on pod yield and economic return were assessed. Genotypes C-11-2-39 and Tifrunner demonstrated the best field resistance to TSWV, whereas cvs. DP-1 and GA-01R and line C-28-305 were among the genotypes with the best leaf spot resistance. Epidemics of both diseases were comparable or suppressed in strip-tilled plots compared with conventionally tilled plots. Leaf spot intensity decreased with increased fungicide applications, but to a lesser degree with use of resistance and strip tillage. Yields and net returns were similar between tillage treatments in 2002 and lower in strip tillage in 2003. Genotypes with the greatest yields and returns were C-11-2-39, C-99R, and GA-01R. Returns were comparable among the four-, five-, and seven-spray programs in both years, despite differences in yield. The standard production system, Georgia Green in conventional tillage with seven sprays, resulted in lower returns than half the integrated systems tested in 2002, but had comparable or higher returns than nearly all systems in 2003. When significant, yields and returns were correlated with spotted wilt intensity to a greater degree than leaf spot intensity.

Effects of reduced tillage, resistant cultivars, and reduced fungicide inputs on progress of early leaf spot of peanut (Arachis hypogaea)

Peanut is vulnerable to a range of foliar diseases such as spotted wilt caused by Tomato spotted wilt virus (TSWV), early (Cercospora arachidicola) and late (Cercosporidium personatum) leaf spots, southern stem rot (Sclerotium rolfsii), and sclerotinia blight (Sclerotinia minor). In this study, we report the generation of 17,376 peanut expressed sequence tags (ESTs) from leaf tissues of a peanut cultivar (Tifrunner, resistant to TSWV and leaf spots) and a breeding line (GT-C20, susceptible to TSWV and leaf spots). After trimming vector and discarding low quality sequences, a total of 14,432 high-quality ESTs were selected for further analysis and deposition to GenBank. Sequence clustering resulted in 6,888 unique ESTs composed of 1,703 tentative consensus (TCs) sequences and 5185 singletons. A large number of ESTs (5717) representing genes of unknown functions were also identified. Among the unique sequences, there were 856 EST-SSRs identified. A total of 290 new EST-based SSR markers were developed and examined for amplification and polymorphism in cultivated peanut and wild species. Resequencing information of selected amplified alleles revealed that allelic diversity could be attributed mainly to differences in repeat type and length in the SSR regions. In addition, a few additional INDEL mutations and substitutions were observed in the regions flanking the microsatellite regions. In addition, some defense-related transcripts were also identified, such as putative oxalate oxidase (EU024476) and NBS-LRR domains. EST data in this study have provided a new source of information for gene discovery and development of SSR markers in cultivated peanut. A total of 16931 ESTs have been deposited to the NCBI GenBank database with accession numbers ES751523 to ES768453.

Disease progress of tomato spotted wilt virus in selected peanut cultivars and advanced breeding lines

BackgroundPeanut is one of the major source for human consumption worldwide and its seed contain approximately 50% oil. Improvement of oil content and quality traits (high oleic and low linoleic acid) in peanut could be accelerated by exploiting linked markers through molecular breeding. The objective of this study was to identify QTLs associated with oil content, and estimate relative contribution of FAD2 genes (ahFAD2A and ahFAD2B) to oil quality traits in two recombinant inbred line (RIL) populations.ResultsImproved genetic linkage maps were developed for S-population (SunOleic 97R × NC94022) with 206 (1780.6 cM) and T-population (Tifrunner × GT-C20) with 378 (2487.4 cM) marker loci. A total of 6 and 9 QTLs controlling oil content were identified in the S- and T-population, respectively. The contribution of each QTL towards oil content variation ranged from 3.07 to 10.23% in the S-population and from 3.93 to 14.07% in the T-population. The mapping positions for ahFAD2A (A sub-genome) and ahFAD2B (B sub-genome) genes were assigned on a09 and b09 linkage groups. The ahFAD2B gene (26.54%, 25.59% and 41.02% PVE) had higher phenotypic effect on oleic acid (C18:1), linoleic acid (C18:2), and oleic/linoleic acid ratio (O/L ratio) than ahFAD2A gene (8.08%, 6.86% and 3.78% PVE). The FAD2 genes had no effect on oil content. This study identified a total of 78 main-effect QTLs (M-QTLs) with up to 42.33% phenotypic variation (PVE) and 10 epistatic QTLs (E-QTLs) up to 3.31% PVE for oil content and quality traits.ConclusionsA total of 78 main-effect QTLs (M-QTLs) and 10 E-QTLs have been detected for oil content and oil quality traits. One major QTL (more than 10% PVE) was identified in both the populations for oil content with source alleles from NC94022 and GT-C20 parental genotypes. FAD2 genes showed high effect for oleic acid (C18:1), linoleic acid (C18:2), and O/L ratio while no effect on total oil content. The information on phenotypic effect of FAD2 genes for oleic acid, linoleic acid and O/L ratio, and oil content will be applied in breeding selection.