Tomato Research Report 2010-2011 - University of Florida

Supported by the Florida Tomato Committee

Tomato Research

Report 2010-2011

Contents

Research supported by the Florida Tomato Committee 2010-2011 IFAS Research Report

Page Title Investigator

Variety Development

1 Breeding Tomatoes for Florida John W. ScottGary E. Vallad

Crop Protection Technologies

6 Development of a Real-Time Polymerase Chain Reaction Assay to Detect and Quantify Fusarium Wilt and Fusarium Crown and Root Rot Pathogens in Florida Tomato Production

Gary E. ValladCheng-Hua HuangAndrew W. MacRae

12 Efficacy of Cultivation and Glyphosate Combinations on the Reduction of High Populations of Yellow and Purple Nutsedge in Florida Tomato

Andrew W. MacRaeGary E. Vallad

16 Evaluation of Tomato Yellow Leaf Curl Virus and Fusarium Crown Rot Resistant Tomato Varieties under Commercial Conditions in Southwest Florida

Monica P. Ozores-HamptonEugene McAvoy

Tomato Research Report 2010-2011 1

Breeding Tomatoes for FloridaJohn W. Scott Gary E. Vallad University of Florida, IFAS Gulf Coast Research & Education Center-Balm 14625 CR 672 Wimauma, FL 33598

AbstractFla. 8611 and Fla. 8835 are plum tomato inbreds

with bacterial spot tolerance that are being considered for release as breeding lines along with some newer inbreds. The release of the best lines should be done after fall 2011 trials. Fla. 8704, a hybrid with bacte-rial wilt-tolerant parent Fla. 8626, is being tested on grower farms in fall 2011. Several promising inbreds with tolerance to bacterial wilt with very large, firm fruit were developed, and phase 1 hybrid testing began. Fla. 8806 is a crimson hybrid with outstanding flavor that had significantly greater early yield than Florida 47 and Sanibel in Homestead. It is now in grower trials. Fla. 8735 is a heat-tolerant inbred that is a parent of Fla. 8806 and several other hybrids that have performed well in multiple yield trials. Fla. 8746 is a spotted wilt-resistant hybrid that is now being tested in grower trials. Jointless inbreds with compact growth habits (CGH) were inconsistent, looking good in one trial but not at all good in others. Despite this, other lines are emerging and perhaps some of these will perform more consistently. A new bacterial spot-tolerant inbred, Fla. 8871, looked very nice in the spring and may be our first inbred with commercial parental potential.

Introduction Improved varieties are needed to keep the Florida

tomato industry competitive in a changing world. Im-provements include the areas of yield, pest resistance, and fruit quality. Florida’s geography offers many challenges for tomato improvement, since high tem-peratures limit fruit set and are conducive to disease and insect problems. Although private companies have tomato breeding programs that develop varieties for Florida, these companies do not handle many of the long-term, high-risk projects that could prove valuable in the future. The University of Florida tomato breed-ing program aims at such projects and works in part-nership with the private companies to deliver improved varieties of benefit to the Florida tomato industry.

Much of the groundwork for the development of heat-tolerant varieties was done at the University of

Florida (Scott et al. 1986). ‘Solar Set’ (Scott et al. 1989) was an important commercial, heat tolerant variety for 11 years after it was released. The improved heat toler-ance in ‘Solar Fire’, released in 2003, has been evident on some farms, but sensitivity to bacterial spot that has been carried with resistance to fusarium wilt race 3 has limited its use. Fusarium wilt race 3-resistant varieties are available because of the introgression of a resis-tance gene discovered in a wild species by this program in the 1980s (Scott and Jones 1989; Scott and Jones 1995). Breeding lines with Fusarium crown and root rot resistance were released in 1999 (Scott and Jones 2000) and UF breeding lines are in commercial varieties ‘Se-bring’, ‘Soraya’, and ‘Crown Jewel’. With the impending loss of methyl bromide, these pathogens could become more widespread. Other diseases such as bacterial wilt and spotted wilt occur in Florida but cause far more damage in other regions of the world. If these diseases become more prevalent here, resistant varieties will prove beneficial. Tomato yellow leaf curl virus (TY-LCV) is a serious threat to Florida tomato production (Polston et al. 1999) and some crops have sustained severe losses in past years. Commercial companies have released TYLCV-resistant varieties, but none have yet been widely accepted by Florida growers. Our project has been ongoing since 1990 utilizing different resistance genes derived from the wild species Sola-num chilense. To date we have identified two resistance genes Ty-3 (Ji et al. 2007) and Ty-4 (Ji et al. 2009) and marker assisted selection (MAS) is now being used to more rapidly move these genes into elite inbreds. Fine mapping of Ty-3 has resulted in breeding lines with tiny introgressions (<72kb) that appeared to be rid of the foliar disease susceptibility and fruit set problems as-sociated with Ty-3 breeding lines with larger introgres-sions. Very recently we located a marker linked to the Ty-5 resistance gene from ‘Tyking’, a line that has been used in our resistance program, and MAS can now be done to speed up incorporation of this resistance gene. We are still searching for at least one other resistance gene. Markers linked to resistance genes have been made available to private company tomato breeding programs.

Shipping tomatoes have long met with dissatis-faction in the marketplace and this has been recently expounded on in the book Tomatoland by Barry Esta-brook. Although this book takes an unfair, negative slant on many aspects of the Florida industry, it has prompted unfavorable newspaper reports and overall conveys a negative perception of shipping tomato qual-ity. Essentially, any problems with fruit flavor relate to compromises that are made in providing fruit that will ship well. Solutions to this problem are not simple.


Research is needed to provide tomatoes that will be more acceptable to consumers. On the bright side, Nguyen and Schwartz (1999) and others have shown that lycopene, the red pigment in tomato, has strong antioxidant properties that reduce several cancers. Work in the breeding program has been ongoing for over 25 years with the crimson gene (ogc) that improves internal tomato color and increases lycopene content. Seven breeding lines with this gene have been released to seed companies over the last 14 years. Crimson varieties may be a boon to the Florida industry in the near future and the crimson hybrid Tasti-Lee provides an avenue for Florida growers to gain back market share in the supermarkets from the greenhouse indus-try. Whereas vine ripe harvesting of Tasti-Lee would require more labor than is used for mature-green harvesting, the development of varieties with genetic alteration of plant architecture (compact growth habit or GCH varieties) would provide ground tomatoes that reduce labor costs for staking and harvesting. This pro-duction system may prove to be important for future production in Florida. Objectives of this project were:

1. To develop tomato varieties or breeding lines with resistance to soil-borne pathogens.

2. To develop inbreds and hybrids with tolerance to bacterial spot.

3. To develop improved heat-tolerant tomato in-breds and hybrids.

4. To develop commercially acceptable tomato breeding lines and hybrids resistant to TYLCV or TSWV.

5. To improve fruit quality, postharvest character-istics, and plant type.

Objective 1Methods

Seedlings of hundreds of lines are inoculated with fusarium wilt races 2, fusarium crown rot, verticillium wilt, and bacterial wilt to select resistant plants for field evaluation. A molecular marker is now used for fusar-ium wilt race 3 screening. The lines showing the most promise are tested in yield trials at GCREC, and some are tested at NFREC or Dade County. The best of these are crossed to test hybrids for commercial release. The hybrids are evaluated at GCREC (stage 1), and the best ones are advanced to replicated trials at GCREC (stage 2). The best of these hybrids are then tested off station at IFAS trials and on grower farms (stage 3). This report will highlight primarily some results of stage 2 testing that took place in the last year.

ResultsFla. 8626 is a bacterial wilt-tolerant-inbred with

huge fruit size and several hybrids have been in stage 2 testing. Some have strong vines with some tolerance to bacterial spot. Bruising of fruit has been a problem with most of the hybrids, but Fla. 8704 has been firm and performed well in trials in Dade County, Gadsden County and at GCREC. It will be tested on 4 grower farms this fall. A new group of bacterial wilt-tolerant- inbreds derived from Fla. 8626 and other lines were selected, and these do not have the bruising problem. A new group of inbreds with resistance to fusarium crown rot have been developed, and hybrids derived from these have been in phase 2 testing. QTLs for bacterial spot tolerance are being incorporated into fusarium wilt race 3 inbreds since we have found them to be extra sensitive to bacterial spot. Fla. 630 has race 3 resistance and tolerance to bacterial spot along with a non-blighting (NB) characteristic that we are incor-porating into advanced inbreds from 630. Leaves of NB lines retain their green color despite foliar disease infection and are slow to become necrotic. We hope to find molecular markers linked to NB to facilitate breeding of this trait. NB would be a boon to any fu-ture tomato variety releases.

Objective 2Methods

Bacterial spot breeding lines and experiments are inoculated in the field, but natural infections often provide enough disease pressure. Breeding lines with better attributes are tested in stage 2 inbred trials and crossed to assess hybrid potential. The best hybrids are tested in stage 2 trials. QTLs were evaluated in experiments each season and populations from resis-tant accessions were evaluated in the spring to locate any undiscovered QTLs in order to provide improved resistance.

ResultsFla. 8517 is a plum tomato line that has been used

as a source of resistance in bacterial spot genetic work. A large number of plum lines are being developed from this project, and in spring 2011 three of these did well in a yield trial. This fall these will be compared to plum inbreds Fla. 8611 and Fla. 8835 that were being consid-ered for breeding line release. The most promising of these will likely be released at the end of 2011 or early in 2012. They will provide private breeders material to develop bacterial spot-tolerant plum hybrids. In spring 2011 Fla. 8871 appeared to be bacterial spot tolerant


and had very nice large fruit. If this line continues to perform well it could be our first large-fruited inbred with attributes necessary for making good commercial hybrids with bacterial spot-tolerance. In order to locate additional resistance QTL, hundreds of F2 and F3 selec-tions were made in populations derived from two of our resistance sources. A large number of NB selections (see objective 1) were also made in the fall and spring. NB has also been found in some lines and hybrids de-rived from the bacterial wilt resistance program.

Objective 3Methods

Heat-tolerant (HT) fruit setting ability is being incorporated into all phases of the breeding program. For instance, many lines being developed with bacterial spot tolerance have heat- tolerance as well. Key selec-tion in this project is done for early fruit setting abil-ity in fall crops when there is high temperature stress. Otherwise procedures are similar as those described under objective 1.

ResultsHybrids with Fla. 8735 have shown some potential

in stage 2 testing and will be tested further for possible release after grower testing. Fla. 8806 has Fla. 8735 as a parent, and it had significantly higher early marketable yield than Florida 47 and Sanibel in a grower trial in Dade County. It has very good flavor and interior color due to high lycopene from the crimson gene. It is being tested this fall on grower farms. Several breeding lines with very large fruit have advanced to phase 2 testing. This fall early planting will allow for a good assessment of their heat-tolerant setting ability. Heat-tolerant hybrids with the proper horticultural characteristics should provide Florida growers with varieties that set marketable fruit more reliably under stress conditions such as cool temperatures as well as under high tem-peratures.

Objective 4Methods

Geminivirus resistance. Hundreds of breeding lines from F2 to F12 are inoculated with TYLCV at the seedling stage via viruliferous whiteflies and then rated for disease severity in the field. Once resistance is fixed, generally in the F3 or F4 generation, backcross-ing is done to make further horticultural improve-ments. The best inbreds are also intercrossed and the resultant hybrids then evaluated for possible stage 2

testing. We have been working intensively to locate molecular markers linked to resistance genes as this is key to developing horticulturally acceptable parent lines and hybrids with TYLCV resistance. Since we now have located markers linked to Ty-3, Ty-4, and Ty-5 from ‘Tyking’, MAS is being used instead of using the inoculation procedure above for some of the pedigrees. Backcrossing using the Ty-3 short introgression donor was actively started and two crosses have already been made with over 20 elite breeding lines. Phenotyping has been done on populations segregating for an as yet unknown resistance gene. DNA has been sent to SolCAP, and they will genotype using over 6,000 SNP markers.

Spotted wilt resistance. Selection for Sw-5 and Sw-7 resistance genes is now done with molecular markers that eliminate the need to screen with thrips and po-tentially spread the virus in the west coast growing re-gion. Preliminary data suggested that fruit blotch was reduced or eliminated in Sw-7 varieties, but this needs to be verified. Several hybrids with each gene have been grown recently to evaluate the incidence of fruit blotch, which has been a problem with Sw-5 hybrids under some conditions. There were 70 recombinant inbred lines tested at NFREC for resistance in a replicated trial in spring 2011. As new inbreds are developed that show horticultural promise, they are crossed and the result-ing hybrids are tested for commercial acceptability.

ResultsGeminivirus resistance. Reliable fruit setting and

susceptibility to foliar diseases have been problems in this program. Growers have been disappointed in existing TYLCV-resistant varieties due to linkage drag problems that appear to be gone in the Ty-3 short introgression lines. Future hybrids should have bet-ter horticultural attributes. The SolCAP data should provide a molecular marker linked to our unknown resistance gene and allow for us to use MAS for all our future breeding work on this project.

A big change has been the implementation of MAS that requires no field work for two years while four backcrosses are made using about 20 elite recurrent parent lines. This work should result in the ability to make acceptable hybrids as has already been done with the recurrent parents, but with TYLCV resistance

Spotted wilt resistance. A number of new inbreds with Sw-5 or Sw-7 are under development and are in stage 2 testing. Hybrid Fla. 8746 with Sw-5 is now in phase 3 testing. Unfortunately, there was inadequate fruit blotch to obtain any useful data for the second year in a row. The spring 2011 experiment with the 70


RIL lines at NFREC has delimited the Sw-7 gene from a 20-cM to a 5-cM region. A few breeding lines with Sw-7 had good horticultural type in trials during the last year. Hybrids are now being tested along with some new hybrids with Sw-5.

Objective 5:Methods

Fruit quality and shelf-life are emphasized in all breeding projects. One aspect of fruit quality is the development of high-lycopene varieties by using the crimson (ogc) gene. This gene is widely distributed in the various breeding projects. New inbreds and hybrids are continually being made and evaluated. Another aspect is the incorporation of high sugars from a cherry line into large-fruited lines and in developing improved cherry/grape breeding lines. We are backcrossing the desirable fruity/floral note into the parents of Tasti-Lee to further improve the flavor. This is challenging be-cause expression of this note is subject to environmen-tal variation and only a few lines are stable for good expression. The most interesting lines from all flavor work are tested in flavor trials with USDA cooperator Dr. Elizabeth Baldwin.

A project has been underway for a number of years to develop a superior crimson, good flavored, jointless tomato cultivar or hybrid especially well-suited for Dade County growing conditions. This is a coopera-tive project with Drs. Jim Strobel and Waldy Klassen. A hybrid trial was conducted on a grower farm in Dade County last winter with the goal to find a hybrid that competes with ‘Sanibel’.

Another project is to develop CGH tomatoes that do not require staking by use of the brachytic (br) gene that reduces plant height and increases side shoots. Here we emphasize jointless tomatoes that can be me-chanically harvested. Selections take place at GCREC and in Dade County on a grower farm.

ResultsCrimson hybrid Fla. 8806 is mentioned under

Objective 3. It has very good flavor and has fruit size comparable to varieties that are presently grown in Florida. In the fruity/floral backcrossing, the most recent crosses were advanced to F2 in the spring. This fall large F2 populations will be grown as we have seen that this trait is present in only small percentages of F2 plants. Of the jointless hybrids tested recently, Fla. 8759 has been the most consistent in Dade County and at GCREC. It is not crimson but appears to have a vine comparable to that of Sanibel. Unfortunately, we

had a seed mix for Fla. 8787, a hybrid that did well in Dade over the previous two years, and it will have to be tested again next year. Two jointless inbreds were outstanding in spring breeding plots, and Fla. 8645 looked very good in the spring yield trial that had very poor growing conditions. In the CGH project, a mile-stone was thought to have been reached in spring 2010 with the emergence of Fla. 8834, an F3 line with joint-less stems, an ideal plant habit, and a concentrated set of early, large, smooth, and firm tomatoes. However, this line did not have good fruit size in the fall where another jointless line did look good. Both were in a grower trial in a winter Dade county trial, and neither set much fruit. Thus, a good jointless CGH inbred has still not been obtained. In spring 2011, a couple of related F3 lines looked good, but it remains to be seen if they will do well consistently. Many new F2s will be tested this fall and hopefully perseverance will soon pay off.

Literature CitedJi, Yuanfu, David J. Schuster, and Jay W. Scott. 2007.

Ty-3, a begomovirus resistance locus near the Tomato yellow leaf curl virus resistance locus Ty-1 on chromosome 6 of tomato. Molecular Breeding 20:271-284.

Ji, Yuanfu, Jay W. Scott, David J. Schuster, and Doug-las P. Maxwell. 2009. Molecular Mapping of Ty-4, a New Tomato Yellow Leaf Curl Virus Resistance Locus on Chromosome 3 of Tomato. J. Amer. Soc. Hort. Sci. 134(2):281–288.

Nguyen, M.L. and S.J. Schwartz. 1999. Lycopene: Chemical and biological properties, Food Technol. 53(2): 38-45.

Polston, J.E., R.J. McGovern, L.G. Brown. 1999. Intro-duction of Tomato yellow leaf curl virus in Florida and implications for the spread of this and other geminiviruses of tomato. Plant Dis. 8384-988.

Scott, J. W., and J. P. Jones. 1989. Monogenic resistance in tomato to Fusarium oxysporum f. sp. lycopersici race 3. Euphytica 40:49-53.

Scott, J. W., and John Paul Jones. 1995. Fla. 7547 and Fla. 7481 tomato breeding lines resistant to Fu-sarium oxysporum f. sp. lycopersici races 1, 2, and 3. HortScience 30(3):645-646.

Scott, J. W., and John Paul Jones. 2000. Fla. 7775 and Fla. 7781: Tomato breeding lines resistant to Fusari-um crown and root rot. HortScience 35(6):1183-1184.


Scott, J. W., S. M. Olson, J. J. Bryan, T. K. Howe, P. J. Stoffella, and J. A. Bartz. 1989. Solar Set: A heat tol-erant, fresh market tomato hybrid. Fla. Agric. Expt. Sta. Circ. S-359 10p.

Scott, J. W., R. B. Volin, H. H. Bryan, and S. M. Olson. 1986. Use of hybrids to develop heat tolerant toma-to cultivars. Proc. Fla. State Hort. Soc. 99:311-314.


Development of a Real-Time Polymerase Chain Reaction Assay to Detect and Quantify Fusarium Wilt and Fusarium Crown and Root Rot Pathogens in Florida Tomato ProductionGary E. Vallad Cheng-Hua Huang Andrew W. MacRae University of Florida, IFAS Gulf Coast Research & Education Center-Balm 14625 CR 672 Wimauma, FL 33598

AbstractFusarium wilt and Fusarium crown and root rot,

caused by Fusarium oxysporum f. sp. lycopersici (FOL) and F. oxysporum f. sp. radicis-lycopersici (FORL), re-spectively, are two important diseases of tomato. Morphological characters cannot differentiate the two pathogens. Although pathogenicity and vegeta-tive compatibility tests may identify the two patho-gens, they are time-consuming and cannot quantify inoculum densities. The objectives of this study were to determine a soil DNA extraction method and to develop rapid, sensitive, and quantitative real-time polymerase chain reaction (RT-PCR) assays for these two pathogens. Effects of different mixtures of bead sizes on soil DNA yield were evaluated. A combina-tion of 0.5 g each of 0.1-mm and 0.5-mm silica beads showed the highest DNA yield. Humic substances were efficiently removed from soil DNA extracts using silica purification. The removal of humic substance should reduce false-negative quantification of RT-PCR. A spike test performed by adding different amounts of conidia to soil showed a significant linear relationship between the amount of genomic DNA of FOL detected by RT-PCR and the level of conidia added. This finding suggested that the soil DNA extraction method was efficient. Primers and TaqMan probes were developed based on the sequences of the virulence gene SIX1 and ribosomal DNA intergenic spacer (IGS) region for FOL and FORL, respectively. A RT-PCR assay specific for FOL was successfully developed, whereas the assay for FORL is still in progress. The RT-PCR assay specific for FOL detected genomic DNA of FOL as low as 80 fg (a genomic equivalent to 1.21 single-celled conidia in a

reaction). The assay will give the capability to identify and quantify FOL in soil, leading to the development of disease threshold models to enable tomato growers to make decisions regarding the use of specific cultural and fumigation practices, and cultivar selection to mitigate crop losses.

IntroductionFusarium crown and root rot of tomato is caused

by the soilborne fungus Fusarium oxysporum f. sp. radicis-lycopersici (FORL). The pathogen can infect at least 36 plant species and has been reported in all con-tinents (Menzies et al. 1990, Menzies and Jarvis 1994). Fusarium wilt caused by F. oxysporum f. sp. lycopersici (FOL) is another important disease of tomato. The disease, thriving in warm weather, was first described in England in 1895 and has been found in at least 32 countries (Jones et al. 1991). The two pathogens can be introduced to new tomato-growing regions by means of infected seeds, transplants, and soils (Jarvis 1988, Jones et al. 1991). Since the two pathogens are morpho-logically indistinguishable from each other and from nonpathogenic isolates of F. oxysporum, it is in need of developing molecular assays to rapidly and accurately identify FOL and FORL.

Real-time polymerase chain reaction (RT-PCR) has been used to detect and study phytopathogenic and an-tagonistic fungi, bacteria, mollicutes, and viruses, mak-ing possible an accurate, reliable and high throughput quantification of target organisms in various environ-mental samples, including plant tissues, soil, water, and air (Schena et al. 2004). Fusarium diseases may develop with fewer CFU than the detection threshold of Fusar-ium-selective media (Nishimura 2007), but RT-PCR has the potential for determining the soil inoculum thresh-old levels necessary for the disease development (Cullen et al. 2002). RT-PCR will provide new opportunities for studying diagnosis, inoculum threshold levels, epide-miology, and host-pathogen interactions for FORL and FOL. An important prerequisite for the accuracy of RT-PCR is a soil DNA extraction method to efficiently disrupt chlamydospores that are dormant structures of FORL and FOL in soil. The objectives of this study were to develop a soil DNA extraction method for FORL and FOL and to detect and quantify the two pathogens using RT-PCR.

Materials and MethodsSoil collection and inoculum. Soil samples were

collected from fields with a history of FOL and FORL at the Gulf Coast Research and Education Center.


The soil samples were air-dried and passed through a 2-mm-diameter sieve. The soil was used to make cornmeal-sand medium (Tuite 1969) inoculated with respective isolate I3 of FOL and isolate CL-0601 of FORL for mass production of inoculum. The inoculum was used to extract Fusarium DNA to develop standard curves for RT-PCR analysis below.

Soil DNA extraction. Three replicate subsamples (0.5 g) were taken to 2-ml vials with different sizes of beads (table 1). Soil was suspended in 1-ml CTAB buffer (Li et al. 2008). After briefly vortexing for 1 min, soil suspensions were homogenized in a FastPrep® instru-ment (Savant Instruments, Inc., Holbrook, NY, USA) for 60 seconds at a speed setting of 6.0. The homog-enate was centrifuged at 14,000g for 10 min. The su-pernatant was transferred to a 1.5-ml microcentrifuge tube and mixed with an equal volume of chloroform/isoamyl alcohol (24:1) before centrifugation at 20,000g for 10 min. The supernatant was transferred to a 15-ml tube and then resuspended with 1 ml of silica bind-ing matrix (Carter and Milton 1993). The tube was inverted by hand for 2 min to allow binding of DNA to silica particles and placed in a rack for 10 min. The suspension was removed, and silica particles were washed with 1 ml of 5.5 M guanidine thioyanate. After centrifuging at 14,000g for 1 min, the supernatant was removed. The washing process was repeated until the silica returned to its original color. The pellet was washed with 70% ethanol by centrifugation at 16,000g for 2 min, air-dried, dissolved in 100 μl of DNase- and pyrogen-free water, and inoculated for 5 min at 55°C in a water bath. Finally, soil DNA was eluted by centrifu-gation at 20,000g for 2 min. If silica particles exist in the supernatant, the centrifugation needs to be repeat-ed before storing soil DNA at -20°C. The quantity and quality of soil DNA were determined using a Nano-Drop® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). To evaluate the efficacy of the soil extraction procedure, a spike test was performed by adding different amounts of conidia of FOL (0–3×109 conidia/g soil).

RT-PCR assays. The DNA sequence of SIX1 (Gen-Bank accession numbers GQ268948) was used to de-sign primers and TaqMan Probe for FOL using Primer Expression version 2.0 (Applied Biosystems, Foster City, CA) since this gene is carried by all races of FOL but not found in the other formae speciales and non-pathogenic isolates of F. oxysporum (van der Does et al. 2008). The primers and probe specific for FORL were designed based on the alignment of IGS sequences. RT-PCR was performed in the Bio-Rad CFX96 Real-Time PCR Systems (Bio-Rad, Hercules, CA). A total volume

of 20 μl of the reaction mixture included the following components (final concentration): 1 × SsoFast Probes Supermix (Bio-Rad, Hercules, CA), 0.9 μM forward and reverse primers, 0.2 μM TaqMan probes dual-labeled with the 6-carboxyfluorescein (6-FAM) fluorescent reporter dye and the Iowa Black® fluorescence quencher (Integrated DNA Technologies, Coralville, IA), 2 μl soil DNA template, and PCR-grade water to make up the final volume. Three simultaneous amplifications were performed for each sample to confirm the reproduc-ibility of the results. A negative control using nuclease-free water was substituted for the DNA template. The manufacturer’s recommended universal thermal cycle protocol (Bio-Rad, Hercules, CA) was used for PCR amplification: An initial hot start of 2 min at 95°C was followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Fluorescence emission was measured at 60°C of the annealing and extension phase. Threshold cycles were calculated using the Bio-Rad CFX Manager 2.0 soft-ware. A standard curve was developed by plotting the logarithm of known concentrations (tenfold dilution series from 80 ng to 80 fg/ 20 μl reaction) against the threshold cycle (Cq) values. Cq is defined as a cycle number at which fluorescence has increased above the background. Cq value is inversely related to log of ini-tial DNA concentrations, suggesting that the lower the Cq value, the higher the initial DNA quantity.

ResultsSoil DNA extraction. Extractions with different

beads were performed using the same beating speed and extraction buffer volume. Bead mixtures signifi-cantly affected the quantity of soil DNA and humic substances (Table 1). Bead combinations F–J showed significantly superior yield compared to standard 0.1 mm beads (treatments A and C). Humic substances were co-extracted with soil DNA, and their quantity significantly increased with increasing soil DNA yield. After silica purification, humic substances were con-siderably removed and did not affect RT-PCR. The bead mixture combining 0.5 g each of 0.1-mm and 0.5-mm silica beads was adopted for our soil DNA extraction protocol. In the spike test, a significant linear relation-ship was observed between DNA of FOL and conidia spiked (Fig. 1). With a regression coefficient of 0.965, this would strongly indicate a high degree of efficiency of our soil DNA extraction method and sensitivity of our RT-PCR.

RT-PCR assays. Three primer sets and TaqMan probes were selected to determine their specific-ity and efficacy in RT-PCR for FOL. Primers SIX1-3F (AACCTGGTACCCCGAATCG) and SIX-3R1 (GAGAGT-


GCCCGCCATCTG), TaqMan probe SIX1-3B (6-FAM-ATCGCCTGGGACCTACGATTATTTCGC-Iowa Black) showed consistency in amplification and Cq values. The primers and probe successfully amplified target DNA of FOL, with a PCR efficiency of 104% based on the formula: Efficiency= 10(1/slope) −1, where the slope equals that of the standard curve (Fig. 2). The RT-PCR was highly sensitive, detecting 80 fg of DNA of FOL in a 20 μl reaction.

The alignment of IGS sequences showed promise in designing primers specific for predominant genotypes of FORL in Florida. The development of primer and TaqMan probes specific to FORL is ongoing.

DiscussionIn this study, we developed a soil DNA extraction

procedure for Fusarium pathogens of tomato and a real-time PCR specific for FOL. The development of the two techniques can be used to identify and quantify FOL in soil. For plant disease management, using RT-PCR to quantify inoculum may help tomato growers make de-cisions regarding various cultural and fumigant man-agement practices to mitigate losses. The development of RT-PCR is essential for developing disease threshold models to relate soil levels of FOL or FORL to potential tomato production losses and to better quantify the impact of management strategies on FOL and FORL soil populations.

Soil DNA extraction methods affect the accuracy of RT-PCR to identify and quantify Fusarium pathogens. Although the manufacturer suggested 0.1-mm silica beads for disrupting fungal spores (BioSpec, Bartles-ville, OK), our results showed that a combination of 0.5g each of 0.1-mm and 0.5-mm significantly increased soil DNA yield. A combination of different sizes of silica beads may enhance the chance to disrupt spores in soil microaggregates and in the bulk soil population. Humic substances are main soil inhibitors, inhibiting DNA polymerase activity and reducing PCR efficiency (Arbeli and Fuentes 2007). Without using silica pu-rification, DNA of FOL was not detected in positive controls. However, our soil DNA extraction method with a silica purification procedure resulted in a high PCR efficiency, suggesting that humic acids were signif-icantly removed.

For a sensitive RT-PCR, the chosen PCR probe and primers need to be highly specific because a number of microbes exist in the soil that will be co-extracted with FOL. The specificity of SIX1 has been demonstrated previously, which is specific for FOL (van der Does et al. 2008). In addition, our RT-PCR assay did not amplify

other formae speciales of Fusarium oxysporum including FORL. This finding suggests that false-positive quanti-fication is highly unlikely. Compared to FOL in Florida, FORL has a higher degree of genetic diversity. Without identifying a species-specific locus, it is difficult to de-velop a RT-PCR assay specific for FORL. Although IGS showed polymorphisms between pathogenic Fusarium species of tomato, more representative isolates need to be sequenced for alignment to design primers and probes specific for FORL. Such sequencing efforts are underway to develop primers and probes specific to the predominant FORL groups in FL.

In conclusion, this study has shown that a soil DNA extraction procedure with silica purification can remove humic substances that cause false-negative results due to the inhibition of PCR. Moreover, our TaqMan real-time PCR assay can quantify FOL in soil, making it possible to detect initial inoculum sources. To establish disease risk categories using RT-PCR, soil sampling strategies need to be developed to detect the spatial distribution of Fusarium in a tomato field. In addition, a sampling depth needs to be determined to accurately uncover the distribution of pathogens in the soil profile. Further development of disease thresh-old models for FOL and FORL based on RT-PCR and appropriate soil sampling strategies will assist tomato growers in making disease management decisions based on a given level of the pathogen in the soil and the relative benefit of disease management strategies (cultivar selection, fumigation program, cover crop selection, or soil management) on the pathogen popu-lation.

Literature CitedArbeli, Z., and Fuentes, C. L. 2007. Improved purifica-

tion and PCR amplification of DNA from environ-mental samples. FEMS Microbiol. Lett. 272:269-275.

Carter, M. J., and Milton, I. D. 1993. An inexpensive and simple method for DNA purification on silica particles. Nucl. Acids Res. 21:1044-1044.

Cullen, D. W., Lees, A. K., Toth, I. K., and Duncan, J. M. 2002. Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quanti-tative real-time PCR. Plant Pathol. 51:281-292.

Jarvis, W. R. 1988. Fusarium crown and root rot of tomatoes. Phytoprotection 69:49-64.

Jones, J. B., Jones, J. P., Stal. J. P., and Zitter, T. A. 1991. Compendium of Tomato Diseases. American Phy-topathological Society, St. Paul, MN.


Li, R., Mock, R., Huang, Q., Abad, J., Hartung, J., and Kinard, G. 2008. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detec-tion of diverse plant pathogens. J. Virol. Methods 154:48-55.

Menzies, J. G., and Jarvis, W. R. 1994. The infestation of tomato seed by Fusarium oxysporum f. sp. radicis-lycopersici. Plant Pathol. 43:378-386.

Menzies, J. G., Koch, C., and Seywerd, F. 1990. Addi-tions to the host ranges of Fusarium oxysporum f. sp. radicis-lycopersici. Plant Dis. 74:569-572.

Nishimura, N. 2007. Selective media for Fusarium oxys-porum. J. Gen. Plant Pathol. 73:342-348.

Schena, L., Nigro, F., Ippolito, A., and Gallitelli, D. 2004. Real-time quantitative PCR: a new technol-ogy to detect and study phytopathogenic and an-tagonistic fungi. Eur. J. Plant Pathol. 110:893-908.

Sonoda, R. M. 1976. The occurrence of a Fusarium root rot of tomato in south Florida. Plant Dis. Rep. 60:271-274.

Tuite, J. F. 1969. Plant Pathological Methods: Fungi and Bacteria Burgess Publishing, Minneapolis, MN.

van der Does, H. C., Lievens, B., Claes, L., Houterman, P. M., Cornelissen, B. J. C., and Rep, M. 2008. The presence of a virulence locus discriminates Fu-sarium oxysporum isolates causing tomato wilt from other isolates. Environ. Microbiol. 10:1475-1485.


Table 1. Effect of bead mixtures on DNA yield and humic substancesBead weight (g per tube) DNA yield

(μg per g of dry soil)A320

Treatment 0.1 mm silica

0.5 mm silica

0.5 mm glass

1 mm glass

A 0.75 1.53 f 0.034 e

B 0.75 0.25 2.10 de 0.043 de

C 0.50 1.92 ef 0.048 cde

D 0.50 0.25 2.11 cde 0.051 bcd

E 0.25 0.25 0.25 2.39 cde 0.054 abcd

F 0.25 0.25 0.25 1 bead 2.56 abc 0.061 abc

G 0.50 0.25 0.25 2.41 bcd 0.064 ab

H 0.50 0.25 0.25 1 bead 2.62 ab 0.063 ab

I 0.25 0.25 2.66 ab 0.064 ab

J 0.50 0.50 2.99 a 0.067 a

Fig. 1. Spike test for evaluating soil DNA extraction protocol developed in this study. Various amounts of conidia of Fusarium oxysporum f. sp. lycopersici were spiked into soil before DNA extraction. DNA quantity of the pathogen was determined using real-time PCR targeting SIX1, a virulence gene.


Fig. 2. Real-time polymerase chain reaction (PCR) standard curve of Fusarium oxysporum f. sp. lycopersici DNA extracted from cornmeal-sand medium. Pasteurized field soil with cornmeal was inoculated with isolate I3 of the pathogen, and DNA was extracted at one month after inoculation using the protocol developed in this study. Various amounts of DNA, ranging from 80 fg to 80 ng, were used as standards. Each point represents three replicates.


Efficacy of Cultivation and Glyphosate Combinations on the Reduction of High Populations of Yellow and Purple Nutsedge in Florida TomatoAndrew W. MacRae Gary E. Vallad University of Florida, IFAS Gulf Coast Research & Education Center-Balm 14625 CR 672 Wimauma, FL 33598

AbstractWith the loss of methyl bromide, there will be an

increase in importance of cultural practices in com-mercial tomato production. Implementing additional cultural practices can help maintain the sustainability of methyl bromide alternatives by keeping problem-atic pests to minimal levels. A trial was conducted to determine the best combination of glyphosate and cultivation for fallow season management of yellow and purple nutsedge. Eight cultivation/glyphosate com-binations were tested in combination with two fumi-gant treatments (PicClor 60 at 250 lbs/treated acre and Paladin Pic at 50 gal/treated acre) and a non-fumigated control. At the time of this report, only data from the 7th and 14th days after fumigant application (DAA) had been collected. Paladin Pic provided almost complete control of all nutsedge present in the field at 14 DAA. PicClor 60 provided 84.1% control of nutsedge, aver-aged over all cultivation treatments. Cultivation-only treatments and a single glyphosate application pro-vided no benefit compared to the no cultural treatment plots. The addition of a second glyphosate treatment reduced the number of nutsedge sprouts by 59.7% for 14 DAA counts, compared to the single glyphosate treatment. The most intensive treatment of glyphosate followed by (fb) cultivation fb glyphosate provided the greatest reduction in nutsedge populations. Compared to the no cultural treatment plots, this would be equal to 77.2% control of nutsedge at 14 DAA. This intensive program promotes additional germination through breakage of the chains of tubers via cultivation, which should be followed by glyphosate to kill those tubers that have sprouted.

Introduction and JustificationWith the phase out of methyl bromide and the

increase in price being observed with the limited sup-ply, fumigation is becoming increasingly costly to the grower. To combat this increase in price, many growers are reducing rates and cutting back on the use of a full fumigant system. Use of a reduced system will often be successful in the 1st and 2nd year following years of methyl bromide application. Use of the reduced system can lead to an increase in pest pressures and the sub-sequent failure of the reduced system. Many of these reduced systems have not been tested in areas of heavy pest infestation and thus their future performance is unknown.

There are currently four registered complete fumi-gant systems for use in Florida. One is Methyl Bromide 50:50, the 2nd is Midas 50:50, the 3rd is the 3-Way system, and the 4th is Paladin Pic. We have observed a loss of Fusarium control with the use of Methyl Bromide 50:50 and no longer consider it a sustainable program. The 3-Way system is currently the most economical fumi-gant system and also provides the greatest opportunity for modification to reduce fumigation costs further. The 3-Way uses a combination of three fumigants to achieve control of nematodes, diseases, and weeds. When any one of the products mentioned below is removed from the system, the level of control on one or two of the areas of pests will be reduced. The 3-Way system is composed of three active ingredients:

1. 1,3 Dichloropropene (1,3-D) – Great on nema-todes, little disease control, and some weed control

2. chloropicrin (Pic) – Great on diseases, little nem-atode control, and some weed control

3. either metam sodium or metam potassium (Me-tam) – Great on weeds, good on nematodes, and fair on diseases.

Two of the pests of the greatest concern are yel-low and purple nutsedge. Many of our full fumigant systems are aimed at controlling these problematic pests. The use of a reduced system will eventually lead to an increase in the level of nutsedge species within a field. One of the reduced systems currently being used in tomato production consists of only PicClor 60 (40% 1,3-D and 60% Pic), which only provides fair control of nutsedge species. Over several seasons the population of nutsedge in a field will increase and even with the use of halosulfuron (Sandea) it may become unmanageable. This will require the addition of KPam into the fumigant program at an average cost of $200


per acre. We do not feel confident that the currently registered full fumigant systems, including the 3-Way system, can clean up a field that is infested with either nutsedge species, but rather they can maintain the level low enough in combination with herbicides that yield loss of tomato will be at a minimum.

Purple nutsedge (Cyperus rotundus L.) reproduces primarily through underground vegetative structures called tubers (Smith and Fick 1937). Although viable seed is produced, the tubers are the greatest concern as it relates to control via fumigation. These tubers are produced in chains and show apical dominance with the germination of the two end tubers and any lateral chain tubers (Smith and Fick 1937). If the terminal tubers are separated from the chain then dormancy is broken on the next set of tubers. Any control strategy that looks to eradicate the presence of nutsedge will only be successful if all the tubers are primed for ger-mination. This can be done with extensive cultivation or a combination of glyphosate and cultivation.

Smith and Fick (1937) observed purple nutsedge producing 93% of all tubers within the top 8 inches of soil. This would put the majority of tubers at a depth that can be reached with cultivation. Cultivation alone is not a viable method for control of nutsedge spe-cies. Smith and Mayton (1938) discovered it would take more than 12 months of disking every two weeks to completely eradicate a field of purple nutsedge. Disking every four weeks would result in a population reduc-tion of 70%.

The addition of glyphosate to cultivation would provide the best means of systematic control of nut-sedge species. Webster et al. (2008) showed an applica-tion of glyphosate will prevent formation of 5th and 6th order purple nutsedge tubers, prevention of 4th order tubers was rate dependent. They also determined that an application of glyphosate at 2.6 lbs ae/A would reduced tuber production by 50%. Edenfield et al. (2005) observed a 99.8% reduction in purple nutsedge tuber density after three years of sequential applications of glyphosate. These results don’t relate directly to plas-ticulture systems, since the nutsedge was in competi-tion with cotton throughout the trial but do show the advantage of adding glyphosate into a fallow manage-ment program.

While there is some research on the use of cultiva-tion and herbicides for the control of nutsedge, their interaction with subsequent fumigant application is currently unknown. If the level of nutsedge exceeds 20 plants per square foot, we know that control is usu-ally not successful with the reduced systems. The next

step is what cultural actions can be taken to reduce the population of nutsedge to a manageable level (12 plants per square foot or less). The use of cultivation and herbicides in the fallow period will be more economical than the addition of KPam into the fumigant system. Not only will the nutsedge population be reduced, but other possible problematic broadleaf and grass weeds may also be reduced. With the registration of Paladin in Florida we will have an increase in control costs of annual grasses and broadleaf weeds with the use of herbicide programs. With the recommended ratio of 79% Paladin to 21% chloropicrin, Paladin Pic provides excellent control of nutsedge species but can still be weak on grasses and can miss some broadleaf weeds when in high populations. The use of a cultural pro-gram during the fallow period will greatly enhance the sustainability of this and all fumigant systems.

Objectives1. Determine the right combination of cultivation

and herbicides that will maximize the reduction of the population of nutsedge species.

2. Determine if the reduced fumigant system PicClor 60 will benefit from the use of a fallow cultural program.

3. Determine if a complete fumigant system (Paladin Pic) will still be necessary after a fallow cultural program.

4. Determine if Paladin Pic can be used as a cleanup fumigant system for high nutsedge populations.

5. Determine if the use of herbicides can increase the performance of the reduced fumigant system in combination with a fallow cultural program.

Materials and MethodsA 8/9 acre block at the Gulf Coast Research and

Education Center was selected with a population of nutsedge averaging 40.6 plants per square foot at the time of trial initiation. The experiment consists of 8 cultural programs and 3 fumigant programs, with and without halosulfuron. The trial was initiated on Febru-ary 25th with a complete cultivation of the entire field. The main treatments were eight cultivation and/or herbicide combinations (Table 1). Each cultural practice plot was 75 ft long by 25 ft wide and consisted of four replications randomized within blocks. Within each cultural practice plot there are 3 tomato beds, with these two of beds receiving either PicClor 60 (250 lbs/


treated acre) or Paladin Pic (50 gal/treated acre). The remaining bed is a non-treated control. Each fumigant treatment will be left without a herbicide treatment or treated with halosulfuron (Sandea at 1oz/ treated A). ‘Security 28’ tomato will be transplanted on August 23rd followed by the halosulfuron applications on Sep-tember 6th.

Data to be CollectedCrop Growth Measurements: Data will be collected

throughout the season to assess crop vigor and re-sponse to these treatments. Measurements will include visual estimates of crop injury, rated on a scale of 0 (no crop injury) to 100 (crop death), as well as crop plant height.

Yield Measurements: The crop will be harvested and graded as per commercial production standards for the length of the growing season that the grower harvests to meet market demands.

Weed Efficacy Measurements: Weed populations that escape the control of the fumigants and/or herbi-cides will be assessed. Within each plot, every purple and yellow nutsedge seedling emerging through the plastic (Cyperus sp.) and the annual grasses and broad-leaf weeds emerging from the planting holes will be counted.

Disease Measurements: Disease incidence within each plot will be identified by pathogen responsible to determine if nutsedge density is correlated to disease pressure in tomato.

Preliminary Data CollectedWeed Efficacy Measurements: At the time of this

report, nutsedge shoot emergence through the plastic mulch at 7 and 14 days after fumigation application (DAA) was counted in the plots and was converted to a per-acre basis. At 7 DAA, Paladin Pic provided 100% control of nutsedge in all plots (Table 2). At 14 DAA, some dormant nutsedge tubers began to emerge in a few of the Paladin Pic plots (Table 3). At 7 DAA, Pic-Clor 60 provided an average of 94.6% control across the entire trial, though 7 days later this level of control had dropped to 84.1% as the dormant tubers began to germinate.

Although this trial is in the initial stages of data collection, the trends observed are consistent with cur-rent best management practices for nutsedge. Cultiva-tion alone does little to reduce the nutsedge popula-tion. It is important in managing nutsedge with the combination of glyphosate and cultivation. The most intensive program promotes additional germination

through breakage of the chains of tubers via cultiva-tion ,which should be followed by glyphosate to kill those tubers that have sprouted. This research is still in its early stages, but the importance on instituting a fallow weed management program can be observed from the data collected so far. The sustainability of a PicClor 60-based fumigation program will be helped with the use of off-season nutsedge management. At 14 DAA, the use of PicClor 60 plus the addition of a single glyphosate application provided 63.8% or greater control of nutsedge compared to a single cultivation treatment receiving PicClor 60 in the bed (Table 3). It may still be possible to use Paladin Pic as a “clean-up” treatment for nutsedge species, although more data will be needed to confirm this possibility.

Literature CitedEdenfield, M.W., B.J. Brecke, D.L. Colvin, J.A. Dusky,

and D.G. Shilling. 2005. Purple nutsedge (Cyperus rotundus) control with glyphosate in soybean and cotton. Weed Technol. 19:947-953.

Smith, E.V. and G.L. Fick. 1937. Nut grass eradication studies: I. Relation of the life history of nut grass, Cyperus rotundus L., to possible methods of control. J. Am. Soc. Agron. 29(12):1007-1013.

Smith E.V. and E.L. Mayton. 1938. Nut grass eradica-tion studies: II. The eradication of nut grass, Cype-rus rotundus L., by certain tillage treatments. J. Am. Soc. Agron. 30(1):18-21.

Webster, T.M., T.L. Grey, J.W. Davis, and A.S. Culpep-per. 2008. Glyphosate hinders purple nutsedge (Cyperus rotundus) and yellow nutsedge (Cyperus esculentus) tuber production. Weed Sci. 56:735-742.

CRIS Project Supported – FLA-GCR-005022


Table 1. Cultivation and Herbicide combinations (Main treatments)Trial 1 (H-Block)

Treat. 1 Treat. 2 Treat. 3 Treat. 4 Treat. 5 Treat. 6 Treat. 7 Treat. 8

Feb 25 Cultivation Cultivation Cultivation Cultivation Cultivation Cultivation Cultivation Cultivation

Apr 04 Glyphosate

Apr 08 Cultivation Glyphosate Cultivation Glyphosate

Apr 30 Glyphosate

May 02 Cultivation Cultivation

May 16 Cultivation Glyphosate Glyphosate Cultivation

May 25 Glyphosate

June 23 Cultivation Cultivation Cultivation Cultivation Cultivation Cultivation Cultivation Cultivation

July 22 Fumigate Fumigate Fumigate Fumigate Fumigate Fumigate Fumigate Fumigate

Aug 23 Plant Plant Plant Plant Plant Plant Plant Plant

Sep 6 Herbicide Herbicide Herbicide Herbicide Herbicide Herbicide Herbicide Herbicide

Table 2. Nutsedge count (7 days after fumigation) by cultural treatmentCultural Treatment Nutsedge Count (7 days after fumigation)

Control PicClor 60 Paladin Pic

---------------- shoots/A ----------------

1) cultivate once 99,000 abc 7,200 a 0 a

2) cultivate twice 131,000 a 8,300 a 0 a

3) glyphosate once 113,000 a 1,400 a 0 a

4) glyphosate twice 54,900 bc 0 a 0 a

5) cultivate then glyphosate 73,800 abc 8,000 a 0 a

6) glyphosate then cultivate 103,000 ab 5,600 a 0 a

7) glyphosate then cultivate then glyphosate 44,300 c 2,200 a 0 a

8) no cultural treatment 115,000 a 7,600 a 0 a

Average across fumigant treatments 91,800 5,000 0

Table 3. Nutsedge count (14 days after fumigation) by cultural treatmentCultural Treatment

Nutsedge Count (7 days after fumigation)

Control PicClor 60 Paladin Pic

---------------- shoots/A ----------------

1) cultivate once 461,000 ab 109,000 ab 762 a

2) cultivate twice 475,000 ab 57,100 bc 0 a

3) glyphosate once 482,000 ab 39,400 c 0 a

4) glyphosate twice 199,000 d 13,900 c 0 a

5) cultivate then glyphosate 258,000 cd 33,800 c 1200 a

6) glyphosate then cultivate 381,000 bc 41,500 c 0 a

7) glyphosate then cultivate then glyphosate 123,000 d 24,800 c 0 a

8) no cultural treatment 540,000 a 144,000 a 0 a

Average across fumigant treatments 365,000 57,900 245


Evaluation of Tomato Yellow Leaf Curl Virus and Fusarium Crown Rot Resistant Tomato Varieties under Commercial Conditions in Southwest FloridaMonica P. Ozores-Hampton University of Florida/IFAS Southwest Florida Research & Education Center 2685 SR 29, North Immokalee, FL 34142

Eugene McAvoy Hendry County Extension Service PO Box 68 LaBelle, FL 33975

AbstractTomato yellow leaf curl virus (TYLCV) and Fu-

sarium crown rot (FCR) caused significant yield reduc-tion to the tomato industry in South Florida. Tomato yellow leaf curl virus is induced by a number of closely related begomoviruses transmitted by the whitefly Bemisia tabaci (Gennadius) and Fusarium crown rot is caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL). Two replicated variety trials were conducted in spring 2011 at Estero, FL, with five and one grafted two plant populations TYLCV and five FCR-resistant round tomato varieties entries and compared with the sus-ceptible grower standards. Overall weather conditions during the trials were normal (dry) and lower than usual temperatures. Whitefly and FCR disease pres-sure was low throughout the season. Therefore, there were no TYLCV and FCR symptomatic plants among varieties. In the TYLCV and FCR, trial total marketable yield (all sizes and harvests combined) was from 1,175 to 2,582 and 2,629 to 3,113 boxes/acre, respectively. For the TYLCV trial extra-large fruit at first harvest, total extra-large (all harvests combined), total marketable yield (all harvests and sizes combined) and unmarket-able yields, the best varieties were ‘SVR 200’, ‘Tycoon’ and the BHN833/Tygress grafted combination at stan-dard planting spacing. However, ‘Tycoon’ produced the highest unmarketable yields. For the FCR, yields were greater for ‘BHN 730’ and ‘HMX 8849’ than for the rest of the varieties in total extra-large fruit at first harvest, total extra-large fruit (all harvests combined) and total

marketable harvest (all sizes and harvests combined). However, ‘BHN 730’ and ‘HMX 8849’ also produced the highest total unmarketable fruits.

IntroductionTomato yellow leaf curl virus and Fusarium crown

rot, caused by Fusarium oxysporum f. sp. radicis-lycop-ersici (FORL), are considered by some to be the worst tomato virus and soil fungal disease affecting the tomato industry in South Florida. Both diseases cause a significant yield reduction in tomato production.

Plants infected with the TYLCV virus display stunted growth and flower abortion with early infec-tions resulting in almost no fruit set (Schuster and Stansly 1996). Management of whitefly and TYLCV relies primarily on insecticides and tomato-free plant-ing periods combined with timely crop destruction after harvest (Schuster and Polston 1999). But, whitefly resistance to insecticide(s) is creating an urgent need for alternative management tools such as TYLCV-re-sistant varieties. Tomato yellow leaf curl virus resistant varieties adapted to our needs and environment have already been developed by companies such as Hazera, Harris Moran, Seminis, Syngenta, and Sakata. Some resistant varieties have been evaluated in UF/IFAS tri-als within the previous 10 years (Gilreath et al. 2000, Cushman and Stansly 2006 and Ozores-Hampton et al. 2008 and 2010). Evaluations from the Ozores-Hampton et al. (2008) trial included nine round and two plum tomato cultivars that resulted in very distinctive differ-ences in performance in the field and fruit quality after the postharvest evaluation under a high virus pressure during spring 2007.

Fusarium crown rot has been a serious disease of tomatoes on fumigated soils for the last 20 years, but its biology and control has been studied only for the past 13 years (Jarvis 1998). Chemical control, such as methyl bromide, has limited efficacy and in many instances is not highly effective. The disease, however, can be managed with resistant varieties, but the lack of consistent fruit quality has been a major factor for not adopting TYCLV-resistant varieties by the Florida to-mato industry. Therefore, growers plant these resistant varieties in limited acreages and continue to take the risk of planting susceptible varieties such as FL47. Va-riety evaluations need to be continued as new genetic material becomes available, and additional information is needed on TYCLV and FCR resistance under local conditions as well as horticultural qualities including postharvest properties, since, for many new cultivars, this information is lacking or insufficient. Ongoing variety evaluation provides independent scientific in-


formation to be able to deliver updated variety recom-mendations other than those suggested by commercial breeding programs. The objective of this study was to document the TYLCV and FCR resistance and horti-cultural characteristics of currently available resistant tomato varieties under commercial field conditions.

Materials and MethodsTwo independent TYLCV- and FCR-resistant vari-

ety trials were conducted and varieties were evaluated in a completely randomized experimental design with four replications during the spring 2011 (Table 1). The trials were located on a tomato farm under commercial growing conditions in Estero, FL. A field with a his-tory of FCR was used for this evaluation. Five and one grafted at two plant populations TYLCV and five FCR-resistant round tomato varieties entries were evaluated and compared with the susceptible grower standards FL 47 and Sebring for the TYLCV and FL 47 for FCR trial (Table 1).

Cultural Practices. Seed were planted in flats and grown by Redi Plants, Corp. The field was rototilled, and the pre-plant fertilizer (bottom mix and top “hot” mix) was applied following the modified broadcast method to supply 300-60-462 lb.acre-1 of N-P2O5-K2O (1 acre = 7,260 linear bed feet). In each trial, tomatoes were grown following industry standards for produc-tion practices (Table 2) and pesticide applications were made as needed in response to regular scouting reports according to UF/IFAS recommendations (Olson et al. 2007). Plant population was approx. 3,967 plants/acre for both the TYLCV and FRC trials. Each tomato va-riety was pruned following the seed company’s speci-fications (Table 1). The field was seepage irrigated, and tomato plants staked and tied.

In both trials, tomatoes were harvested at the mature green stage three times (Table 2) and graded in the field using a potable grading table according to USDA specifications for extra-large (5x6), large (6x6), and medium (6x7) fruit categories (USDA 1997). For the TYLCV trial, total unmarketable tomato fruit numbers and weight were recorded and categorized into blos-som end scar (BES), zipper (Z), odd-shape (OS), scarring (SC) and cracking (CR) described by Ozores-Hampton et al. (2008). Tomato yellow leaf curl virus- and FCR- symptomatic plants were counted at the third harvest on May 4 for both the TYLCV and FCR trials. The number of plants showing symptoms of bacterial spot and speck (caused by Xanthomonas perforans and Pseu-domonas syringae pv. Tomato, respectively) was rated as percentage foliar cover (disease severity) at the third harvest on May 4 for both trials.

For post-harvest evaluation, tomatoes were har-vested at the green mature stage on 13 April 2011 (har-vest 1 for both trials). Tomatoes were placed in 25-lb boxes and transported to the Garguilo, Inc. packing-house (Immokalee, FL.). After 9 days of ethylene ripen-ing treatment (full color), a subsample of 10 tomatoes from each variety and replication was transported to the Southwest Florida Research and Education Center/IFAS Vegetable Laboratory in Immokalee, FL. for qual-ity evaluation. Firmness was measured as fruit defor-mation by using an 11-mm probe and 1-kg force applied to the fruit surface of the fruit equator area after 5 seconds of applied force with a portable digital firm-ness tester (IRREC tester; Ritenour et al. 2002). Higher values of fruit deformation indicate softer fruit. Color was measured using a 1-to-10 scale.

Extension Activities: A total of two well-attended field days on 13 April for TYLCV and FRC were held at the cooperating growers farm in Estero, FL. Partici-pants evaluated the varieties using a 1-to-5 scale (1= very poor; 5=very good) in a blind test for a combina-tion of characteristics including earliness, plant vigor, fruit size, firmness, fruit quality, potential yield and an overall plant rating.

Data were analyzed using ANOVA and Duncan’s Multiple Range Test at 5%. Amount of fruit defects by TYLCV, FCR, and bacterial spot percentage by variety were transformed by Arcsin distribution before the ANOVA, Duncan’s Multiple Range Test, and Least Sig-nificant Difference (SAS, 2009).

Results and DiscussionOverall weather conditions during the trials in

Southwest Florida as recorded by the Florida Automat-ed Weather Network (FAWN) were normal (dry) and lower than usual temperatures with two freeze events (13 Jan. and 23 Jan.), with an accumulation of 7.9 inches of rainfall during the spring of 2011.

Tomato yellow leaf curl virus incidence, bacterial spot, and Fusarium crown rot rating: In spring 2011, the whitefly population was low throughout the season. Therefore, there were no TYLCV-symptomatic plants among any of the varieties (Table 1). Disease pressure for bacterial diseases was high in spring 2011. Thus, there were significant differences in the amount of bacterial spot and speck among varieties in the TYLCV and FCR trials (Table 1). The incidence of bacterial spot and speck ranged from 45 and 77% (TYLCV) and 41 and 57% (FCR). Among the TYLCV and FCR variet-ies, the most susceptible to bacterial spot and bacterial speck were ‘Tycoon’ and ‘Soraya’ as compared to FL 47,


respectively. There were no FCR-symptomatic plants among varieties in either TYLCV or FCR trials.

Fruit yields: First harvest of extra-large fruit ranged from 665 to 1,632 boxes/ acre (TYLCV) and 1,137 to 1,653 boxes/acre (FCR), total extra-large (all harvests com-bined) 781 to 1,951 boxes/acre (TYLCV) and 1,513 to 2,222 boxes/acre (FCR), and total marketable yield (all sizes and harvests combined) from 1,175 to 2,582 boxes/acre (TYLCV) and 2,629 to 3,113 boxes/acre (FCR) (Tables 3).

TYLCV 2011: Based on extra-large fruit at first harvest, total extra-large (all harvests combined), total marketable yield (all harvests and sizes combined) and unmarketable yields the best varieties were ‘SVR 200’, ‘Tycoon’ and the BHN833/Tygress grafted combination at standard planting spacing (P≤0.05, Table 3). Howev-er, ‘Tycoon’ produced the highest unmarketable yields (Table 3). The lowest marketable yields were produced by the BHN833/Tygress grafted combination under a low density planting spacing. The most common defect types as percentage of the total unmarketable yields were BES ranging from 0 to 52.1%, Z ranging from 12.9 to 61.6%, OS ranging from 5.5 to 25.2%, SC rang-ing from 16.0 to 27.5 and CR ranging from 1.7 to 28.2% (data not shown). The large amount of unmarketable (BES, Z, OS, SC and CR) fruit found with TYLCV-resistant varieties during this season was lower than results from spring 2007 and 2008 (Ozores et al. 2008) and consisted with other studies (Gilreath et al. 2000; Scott, 2004 and Cushman and Stansly 2006).

FCR 2011: Yields were greater for ‘BHN 730’ and ‘HMX 8849’ than for the rest of the varieties in total extra-large fruit at first harvest, total extra-large fruit (all harvests combined) and total marketable harvest (all sizes and harvests combined (P≤0.05, Table 3). However, ‘BHN 730’ and ‘HMX 8849’ also produced the highest total unmarketable fruits (Table 3).

Post-harvest and blind test evaluation: There were no differences among TYLCV varieties on fruit firm-ness (Table 4). The highest color rating was that of ‘Charger’ and the lowest ‘Tycoon’, ‘Tygress’, ‘grafted combination BHN833/Tygress’ standard density and ‘Sebring’ (Table 4). Among the FCR-resistant varieties, the highest firmness was from ‘Soraya’ and ‘Sebring’ and the softer fruit was ‘Crown Jewel’. The highest color rating was that of ‘FL 47’ and the lowest ‘Soraya’ during spring 2011.

The extension activities: A field day held at the cooperating grower’s farm in Estero was attended by 56 people. According to 21 (TYLCV) and 17 (FRC) re-sponses from participants in the blind test, the highest overall rating was given to the grafted variety ‘BHN833/

Tygress’, as well as ‘Charger’, ‘RFT80773’, ‘FL 47’ and ‘Sebring’ as compared to all other TYLCV variet-ies (P≤0.01, Table 4). A blind test comparison among FCR varieties indicated that ‘BHN 730’, ‘HMX 8849’ and ‘Soraya’ rated higher than the rest of the varieties (P≤0.01, Table 4).

The authors wish to thank the Florida Tomato Committee, BHN, Harris Moran, Hazera, Sakata, Seminis, Syngenta, Redi-Plants, and Six L’s Farms for providing monetary or in-kind support to these projects.

ReferencesCushman, K. and P. A. Stansly. 2006. TYCLV-resistant

tomato cultivar trial and whitefly control. Proceed-ings: Florida Tomato Institute. P. Gilreath [Ed.], Vegetable Crops Special Series, IFAS, U. of Florida, Gainesville, pp. 29-34.

Gilreath, P., K. Shuler, J. Polston, T. Sherwood, G. McAvoy, P. Stansly, and E. Waldo. 2000. Tomato yellow leaf curl virus resistant tomato variety trials. Proc. Fla. State Hort. Soc. 113:190-193.

Jarvis, WR. 1998. Fusarium crown and root rot of to-matoes. Phytoprotection. Vol.69, no. 2, pp. 49-64.

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Table 1. Pruning, TYLCV virus incidence, bacterial spot, and Fusarium crown rot of Tomato Yellow Leaf Curl Virus and Fusarium Crown Rot resistant tomato varieties evaluation during spring 2011, Estero, FL.

Variety Source PruningNo Suckers

Virus incidence (%)

Bacterial Spot Ratingz (%)

Fusarium crown roty (%)

TYLCV Spring 2011

Charger Sakata No 0 65.0bx 0

RFT 80773 Syngenta 3 0 45.0d 0

SVR 200 Seminis 3 0 55.0bcd 0

Tycoon Hazera 3 0 77.5a 0

Tygress Seminis 3 0 55.0bcd 0

BHN 833/Tygress BHN/Seminis 3 0 52.5cd 0

BHN 833/Tygress LD BHN/Seminis 3 0 55.0bcd 0

FL 47 Seminis 3 0 57.5bc 0

Sebring Syngenta. 3 0 62.5bc 0

P value - 0.0001 -

Sig. - ** -

FCR Spring 2011

BHN 585 BHN 3 0 50.0abx 0b

BHN 730 BHN 3 0 50.0ab 0b

Crown Jewel Seminis 3 0 50.0ab 0b

HMX 8849 Harris Moran 3 0 50.0ab 0b

Soraya Syngenta 3 0 57.5a 0b

FL 47 Seminis 3 0.3 41.0b 1.3a

Sebring Syngenta 3 0 45.0b 0b

P value - 0.01 0.0001

Sig. - ** **

LD = Low plant density. zBacterial spot assessment as disease severity (percentage symptomatic tissue). The rating did not distinguish between the bacterial spot caused by Xanthomonas perforans and bacterial speck caused by Pseudomonas syringae pv. tomato. yFusarium crown rot (Fusarium oxysporum f.sp.radicis-lycopersici). xMeans separation by Duncan’s Multiple Range Test at P ≤ 0.05 Level, and LSD (for FCR data) at the P level in table, means followed by the same letter are not statistically different ** Significance at P ≤ 0.01; * Significance at P ≤ 0.05; ns Not significance.


Table 3. First, total marketable and unmarketable (cull) fruit yield categories for Tomato Yellow Leaf Curl Virus and Fusarium Crown Rot resistant tomato varieties during spring 2011, Estero, FL.

Yield (boxesz/acre)

First Harvest Total Harvest

XLy Ly My FHT XL L M Cull Total

TYLCV Spring 2011

Charger 1,274bx 257ab 85 1,617a 1,503c 459abc 376ab 998ab 2,338a

RFT80773 1,343ab 272ab 118 1,733a 1,607bc 455abc 264abcd 778bc 2,326a

SVR200 1,587a 200bc 91 1,878a 1,780abc 356c 249cd 624c 2,385a

Tycoon 1,632a 201bc 89 1,921a 1,951a 376c 255bcd 1,181a 2,582a

Tygress (TY) 1,395ab 263ab 61 1,719a 1,622bc 433bc 256bcd 527cd 2,311a

BHN833/TY 1,616a 235abc 83 1,934a 1,834ab 415c 271abcd 497cd 2,519a

BHN833/TY LDY 665c 147c 61 872b 781d 238d 157d 305d 1,175b

FL 47 1,364ab 302a 115 1,780a 1,708abc 536ab 319abc 545cd 2,563a

Sebring 1,185b 306a 125 1,617a 1,550bc 539a 382a 522cd 2,471a

P value 0.0001 0.01 0.24 0.0001 0.0001 0.0001 0.007 0.0001 0.0001

Sign. ** ** Ns ** ** ** ** ** **

FCR Spring 2011

BHN 585 1,225b 409a 172ab 1,805ab 1,576b 744a 563a 494b 2,883abc

BHN 730 1,503a 300c 155ab 1,957a 2,076a 638a 400b 891a 3,113a

Crown Jewel 1,183b 319bc 137ab 1,639b 1,513b 667a 449b 725a 2,629c

HMX 8849 1,653a 196d 58c 1,907a 2,222a 494b 281c 750a 2,996ab

Soraya 1,231b 293c 124bc 1,647b 1,697b 662a 373bc 503b 2,731bc

FL 47 1,190b 411a 191ab 1,793ab 1,705b 734a 420b 478b 2,859abc

Sebring 1,137b 403ab 200a 1,740ab 1,650b 781a 369bc 477b 2,800bc

P value 0.0001 0.0003 0.004 0.04 0.0001 0.006 0.0007 0.0001 0.02

Sig. ** ** ** * ** ** ** ** *z25-lb tomatoes/boxyLD = Low plant density; XL= Extra-large (5x6 industry grade); L=Large (6x6); M=Medium (6x7)xMeans separation by Duncan’s Multiple Range Test, P ≤ 0.05 Level, means followed by the same letter are not statistically different. ** Significance at P ≤ 0.01; * Significance at P ≤ 0.05; ns Not significance.

Table 2. Summary of cultural practices used in Tomato Yellow Leaf Curl Virus and Fusarium Crown Rot resistant varieties during spring 2011, Estero, FL.Cultural practice TYLCV 2011 FCR 2011

Plant spacing (inch) 22 22

Bed spacing (feet) 6 6

Bed width (inch) 32 32

MeBr:Chloropicrin (50:50)(lb/acre) 100 100

Mulch Black Black

Planted length (feet) 37 (20 plants) 37 (20 plants)

Harvest length (feet) 18 (10 plants) 18 (10 plants)

Replications 4 4

Transplant date 7 Jan. 2011 7 Jan. 2011

Harvest dates 13 April26 April

4 May 2011

13 April27 April

4 May 2011


Table 4. Post-harvest firmness (measured as fruit deformation), color of fruits at full red stage from first harvest and blind evaluation of tomato plant and fruit characteristics (from the contribution of 21 and 17 participants for Tomato Yellow Leaf Curl Virus and Fusarium Crown Rot resistant tomato varieties during spring 2011, Estero, FL. (Blind evaluation based in rating scale 1-5; 1= very poor and 5 = very good).Varieties Post-harvest Blind Evaluation

Deformationz (mm) Colory (1-10) Overall Rating (1-5)

TYLCV Spring 2011

Charger 2.98 7.0ax 3.3ab

RFT80773 3.03 6.0b 3.1abc

SVR200 2.97 6.0b 2.8bcd

Tycoon 3.40 5.0c 2.4d

Tygress (TY) 3.10 5.0c 2.7cd

BHN 833/TY 3.21 5.0c 3.4a

BHN 833/TY LD 2.98 6.0b 2.9abc

FL 47 2.62 6.0b 3.4a

Sebring 2.01 5.0c 3.2abc

P value 0.08 0.0001 0.0001

Sign. Ns ** **

FCR Spring 2011

BHN 585 3.26b 6.0b 2.9b

BHN 730 3.62b 6.0b 3.5a

Crown Jewel 4.80a 6.0b 2.8b

HMX 8849 3.19b 6.0b 3.4a

Soraya 2.20c 5.0c 3.1ab

FL 47 3.29b 7.0a 2.9b

Sebring 2.04c 6.0b 2.9b

P value 0.0001 0.0001 0.001

Sig. ** ** **zVery firm ≤ 0.7 mm; firm ≤ 1.4mm; medium ≤ 2.1 mm; Soft ≤ 2.8 mm; very soft ≥ 3.9 mm.y1= green and 10 = purple.xMeans separation by Duncan’s Multiple Range Test, P ≤ 0.05 Level, means followed by the same letter are not statistically different ** Significance at P ≤ 0.01; * Significance at P ≤ 0.05;

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Tomato Research Report 2010-2011 - University of Florida