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Deployment Considerations for Late Blight and Early Blight Resistances in TomatoesMartha A. Mutschler and Min-Jea
Kim and Thomas A. Zitter |
Overview of Project: The objectives of the work in the first year were to:
Meeting these objectives was attempted through a set of field experiments located in New York. A series of tomato genotypes were generated that represented homozygous, heterozygous and fully susceptible lines for early blight (Table 1) The plant also ranged from early to late in maturity.
Table 1. The Tomato Genotypes used for Early Blight Trials in Year 1.
|
Pedigrees |
Expected reaction and line Maturity |
|
1. Piedmont |
Early blight susceptible, late maturity |
|
2. NC96LB |
Homozygous EBR, late maturity |
|
3. Piedmont X NC96LB |
Heterozygous EBR late maturity |
|
4. NC215E |
Homozygous. EBR, late maturity |
|
5. CULBPTA46-2. |
Early blight susceptible, early maturity |
|
6. NC96LB X CULBPTA46-2. |
Heterozygous EBR, midseason maturity |
|
7. NC215E X NC96LB |
Homozygous EBR, late maturity |
|
8. NC215E X CULBPTA46-2. |
Heterozygous EBR, midseason maturity |
Establishing the field: Four-week-old plants were grown in 72 cell Speedling under greenhouse conditions until ca. 4 weeks of age, then acclimated to outdoors in a cold frame for a few days. The plants were transplanted on 20 June 2002 into a field with Howard gravelly loam at the H. C. Thompson Research Farm, Freeville, NY. The four main tomato genotypes (1-4 in Table 1) were arranged in a randomized complete block design with four replications. The main plot fungicide treatments applied to these lines were the control (water), a biological fungicide Sonata (Bacillus pumilus), and the alternation of the strobilurin fungicide Cabrio (pyraclostrobin) and Sonata. 4 replications of tomato lines 5-8 (Table 1) were also included in the control (water) plot. Within this plan, each block consisted of eight plants spaced 3 ft. apart within a 24-ft row and 6 ft apart from the susceptible spreader row with 12 ft between each block. A susceptible tomato spreader row was included on one side of the test rows with the test genotypes, providing a constant disease source and pressure.
Spray application: Fungicides were applied with a CO2 pressurized boom sprayer at 40 psi, delivering 15.5 gal/A through four TeeJet XR11003 flat fan nozzles spaced 20 in. apart. The control was a water only spray. Sprays were applied on a 7-day schedule (July 18 and 25; August 1, 8, 21, 27; and September 4). Sonata was applied at the rate of 2% v/v, and Cabrio at the rate of 12 oz/A (0.75 lb/A or 0.15 lb ai).
Inoculation: The spreader rows were inoculated with 15,000 spores/ml of A. tomatophila, 10ml per plant on 19 Jul. The Alternaria strain used in Early Blight evaluation in the first year was the dark strain of Alternaria tomatophila. Rainfall (in. ) was 7.01, 1.28, 2.38, 6.32 for June, July, August and September, respectively, and was supplemented with overhead irrigation.
Data collection: Defoliation was assessed using the Horsfall-Barratt rating scale (0-11) on 6, 13, 19, 26 Aug; and 3, 9, 19, 30 Sep for early blight symptoms and then converted to percent defoliation with Elanco conversion table. Foliar data were calculated using the area under the disease progress curve (AUDPC) model to account for foliar disease progress over time. The data were analyzed using one-way ANOVA at P=0.05 and significant differences between means were separated using Tukey test at P=0.05.
An unusually wet spring delayed our transplanting by nearly two weeks, followed by extremely hot, dry weather during July and August. Both factors had the effect of minimizing disease establishment and spread until late in the season. Early blight appeared on the lower leaves by August 6, which is almost three weeks later than the usual history of this experimental area. The planned August 15 spray was eliminated in order to encourage early blight development. This strategy proved effective. Although the weather conditions delayed the planned data collections, the disease pressure obtained later in 2002 was very severe. Meaningful separation among the treatments was apparent by the September 9 ratings, one week after the final spray date. Disease ratings were taken until September 30.
1. Test of four genotypes with Sonata, Sonata/Cabrio and water control treatments: One section of the trials involved the effects of sonata, an alternating program of Sonata and Cabrio, compared with a water control, against four fresh market tomato genotypes (Figure1). The genotypes, numbers 1 to 4 in the table above, consisted of a line homozygous for early blight resistance (NC215E), a line resistant to both EB and LB (NC96LB), a susceptible line (Piedmont) and the cross of the last two lines, yielding a genotype heterozygous for EB and LB resistance. These entries are all fairly closely related, with Piedmont being a parent in the pedigree of NC215E, and NC215E being a parent in the pedigree of NC96LB. This allows a finer focus of the effects of the resistance genes in these genotypes on disease response.
The susceptible control, Piedmont, had significantly more defoliation than these two resistant lines in the water control (Fig.1a). Disease on the control was uniform, and the susceptible controls in the water control treatment ended the season with ca. 50% defoliation, an indication of the severity of the trial.
The lowest levels of early blight occurred in lines with early blight resistance in a homozygous background, represented by NC96LB and NC215E. These two genotypes performed indistinguishably under all three treatments; there is no indication that the presence of the LB resistance either reduces or increases early blight disease resistance. It is important to note that this resistance does not completely block the disease under severe conditions in the water control treatment, however the cumulative disease of these resistant genotypes was ca. half that of the susceptible control in the absence of applied control agents. The resistance could be a valuable part of a total IPM strategy for early blight control.
Under the severe conditions present in this field trial, early blight resistance expressed in a heterozygous resistant genotype was not significantly different than the fully susceptible line, Piedmont, with the absence of any other control agents (water control). Therefore, the resistance must be used in a homozygous state in order for early blight resistance to be maximally effective under severe disease pressure.
Sonata did have some effect on disease development (Fig.1b). In the Sonata treatment, the two homozygous resistant genotypes had lower cumulative disease development than the heterozygous and susceptible genotypes, but these differences were not longer statistically significant. This was due to reduction of disease development in the heterozygous resistant and susceptible genotypes treated with Sonata compared with these genotypes in the water control. Despite the fact that Sonata used alone provided better control than the water check for the susceptible genotype, this treatment still ended the season with over 25% defoliation in the fully susceptible control. Therefore Sonata alone did not provide satisfactory protection. There is no indication that Sonata reduced disease development in the resistant genotypes, limiting its utility in an IPM strategy using plant resistance combined with other controls.
The treatment alternating Cabrio/Sonata treatments provided excellent control of disease on all tomato genotypes (Fig 1c). Since Sonata alone provided limited control, the effect of Cabrio/Sonata combination could be due to Cabrio alone, rather than the combination. The control of the disease by Cabrio/Sonata was sufficiently good, that it was not possible to determine if resistance provided further protection. However the combination could theoretically reduce the likelihood of the pathogen developing resistance to the chemicals or the resistance genes in the plants, since both would have to occur for the pathogen to thrive in the presence of both forms of control.
2. Alternative genotypes varying for maturity:Additional plant genotypes (items 5 to 8 in Table 1) were added as an extension of the trials using no chemical treatments (Figure 2). One added genotype (number 7 in Table 1) was the cross of two early blight resistant fresh market tomato lines, one of which is also resistant to late blight. The resulting cross is homozygous for early blight and heterozygous for late blight resistance. The data indicate that there was no significant advantage or disadvantage to either homozygosity or heterozygosity for late blight resistance for the control of early blight. (Fig 2a)
The other additional crosses (items 5, 6 and 8 in Table 1) were included to determine the effects of plant morphology and maturity on expression of early blight. Line 5, which is a parent in the crosses that are genotypes 6 and 8, is a processing tomato line that does not carry early blight resistance. Processing tomatoes differ from fresh market tomatoes in a number of ways, including marked differences in plant structure. A processing tomato plant will be more highly branched and branch earlier in development than a fresh market tomato, resulting in a simultaneous development of more branches and flower clusters. This gives the processing tomato its concentrated fruit set. It also quite possibly imposes a greater stress on the plants, since they are supporting the demands of a heavier fruit load earlier in development. This could affect the development of diseases, such as early blight, that tend to have greater affects in stressed plants. The processing tomato line included was also of earlier maturity than the fresh market materials used. Earlier lines can also appear to be affected earlier or to a greater extent than later maturity lines.
The disease development observed in the early maturity processing tomato line was substantially greater than that in the late maturity fresh market line. The processing tomato line was over 60 % defoliated by the end of the season, while the susceptible fresh market line was less than 50% defoliated by the end of the season. Early blight is not considered a major disease in the major processing tomato region (California) largely due to conditions that are generally not conducive to the disease. It can be a problem regionally within the west coast production area, in the eastern production area (Ohio). Early blight damage can also be considerable in hybrid tomato seed producing regions. The results of this trial indicated that under the right conditions, processing tomatoes can suffer great damage due to early blight, and that transfer of the early blight resistance to processing tomato could be an advantage.
Both of the hybrids between an Early Blight susceptible processing tomato line and a homozygous early blight resistant fresh market tomato (genotypes 6 and 8) developed substantially more disease than the homozygous resistant parent Fig 2b and 2c). In the case of genotype 6, the hybrid was fully as susceptible as the resistant parent, approaching 70% defoliation by the end of the season. In the case of genotype 8, the hybrid was more resistant than the susceptible parent, but was still over 60% defoliated by the end of the season. Clearly the presence of the resistance in the heterozygous condition is not adequately protecting these genotypes.
Summary of Early Blight trials in year 1:
These results of these trials were immediately shared with seed companies developing resistant tomato varieties, with the strongest suggestion that new varieties in development should be homozygous for the resistance. This information was critical to the companies, since prior expectations had been that heterozygous resistance would be sufficient for disease control.
The information was also presented at meetings of extension agents and university and USDA associated plant pathologists and plant breeders, to inform them of the use of the control agents in the presence and absence of Early Blight resistance.
2. Towards Deployment of Genetic Resistance for the Control of Tomato Late Blight – 2002 season
Overview of Project: The objectives of the work in the first year were to:
A. 2002 Late blight Field tests of the Utility of Current forms of Late Blight resistance
Two sets of genotypes were used in these trials. The genotypes for the first set of crosses (A), which were exposed to three control treatments, were chosen to provide contrasts in the resistance genes and maturity of the plants (Table 2). All of the parents were processing tomato lines. The parental lines were either homozygous for the presence of the late blight resistance derived from the wild tomato L. pimpinelifolium L3708 (the two Cornell breeding lines) or for the absence of the early blight resistance gene (E6203), and so produce hybrids heterozygous for this form of late blight resistance. The two Cornell lines differed considerably in maturity, either early or late, and the hybrids of E6203 crossed with these two Cornell lines are of mid and late maturity, respectively. None of these lines or hybrids has Early Blight resistance.
The genotypes for the second set of crosses (B), which were not exposed to control chemicals, were chosen to provide contrasts in the resistance genes, maturity and vine types of the plants. The parental lines were either homozygous for the presence of the late blight resistance derived from L. pimpinelifolium L3708 (the early maturity Cornell breeding line and NC 96LB) or for the absence of the late blight resistance gene (NC251E). Another parent (Legend), is homozygous for an alternative late blight resistance gene, Ph2. and so produce hybrids heterozygous for this gene. Legend, NC215E, and NC 96LB are all fresh market tomatoes, and therefore have a larger vine type and less concentration of set than the processing tomato parent. Legend is fairly early, like the Cornell line, but the rest of the parent are late in maturity. Therefore the hybrids vary for maturity as well. The two NC lines also both possess resistance to early blight.
We planned to inoculate all of these plots with the Phytophthora infestans isolate US-17 which can cause disease on plants heterozygous for L3708-derived late blight resistance in lab tests using detached tomato leaves. However there was a problem with the preparation of the US-17 inoculum initially, and at the same time, our plot became contaminated with US-11 when another project applied an early inoculation of that isolate in a neighboring part of the farm. Therefore the experiment was shifted to a test with US-11.
|
A. LB I |
6 genotype, 8 plant each rep, 4 reps |
|
E6203 x 993186-5-2 |
Susceptible E6203 x Early maturity CU LBR line |
|
993181-5-4 X 993186-5-2 |
F1 hybrid between Early maturity CU LBR line and LATE maturity CU LBR line |
|
993186-5-2 |
Early maturity CU LBR line |
|
993181-5-4 |
LATE maturity CU LBR line |
|
E6203 |
Fully susceptible - E6203 |
|
E6203 x 993181-5-4 |
Susceptible E608 x LATE maturity CU LBR line |
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B. LB II |
9 genotype, 8 plant each rep, 4 reps |
|
NC215E X NC96LB |
NC 215E X LB/EB line from NC 96LB |
|
Legend |
Ph-2 source that is determinate |
|
993154-10-3 |
CU A46-derived line - CU early maturity LBR |
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NC215E X 993154-10 |
NC 215E X CU early maturity LBR |
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Legend X NC96LB |
Legend X LB/EB line from NC 96LB |
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NC96LB X 993154-10 |
LB/EB line from NC 96LB X CU early maturity LBR |
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NC215E |
NC215E |
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NC96LB |
LB/EB line from NC 96LB |
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Legend X 993154-10 |
Legend X CU early maturity LBR |
For field plots of both the A and B sets of genotypes, the susceptible rows and “disease spreader” border rows of susceptible plants were evenly and highly diseased due to the incursion of US-11. This shows that infection was uniform in plots even though it was an incursion than a planned application. Both the heterozygous and homozygous resistant genotypes were equally resistant to US11. There were no differences in late blight response of the E6203-derived hybrids vs. the NC215E-derived hybrids, which differ for plant type and maturity, when inoculated with US-11. The only difference in these plots was our inability to score the control rows of E6203 for disease due to late blight, since these controls rows suffered substantial early defoliation due to early blight.
The results of the trials show that the L3708-derived resistance, in the homozygous or heterozygous genotypes, performs very well against US-11 in field trials. This result fully parallels the results obtained in past and current (see below) lab-based tests with this isolate. This is promising, since US-11 is the major isolate in the west coast production areas. However caution is strongly urged in using this resistance in the heterozygous genotype, since the same lab assays with other isolates (such as US-7 and US-17) indicate that resistance to these isolated is only provided by the homozygous genotypes. It is unfortunate that confirmation of this result in field trials could not be obtained this year due to the US-11 incursion.
Typically Ph-2 lines show more disease in field trials than was obtained this year with US-11. The Ph-2 containing line Legend shows very little foliar expression of late blight with US-11, although traces of fruit infection was observed at the end of the season. There was too little infection of the Ph-2 control rows exposed to US-11 for any conclusions regarding the benefits to late blight control though combining Ph-2 with the L3708 derived resistance. Further trials will be needed to address this question.
B. 2002 Lab-based blight tests of the Current Forms of Late Blight resistance
The genotypes included in these tests can be separated into three sections according to the goal of the tests (see Table 3), however the tests were done using the same methods and at the same time. Two isolates were used, US-11, and US-7. The isolates were chosen because US-11 was the isolate that spread into the field trial, and US-7, like US-17, is an isolate in some prior tests could distinguish between homozygous and heterozygous effects. -
A. 3 genotypes, 2 plant each rep, 4 reps, 3 times, Isolate US-11
|
Genotype |
Characteristics of Genotype |
Late Blight Results |
|
E6203 x 993186-5-2 |
Susceptible E6203 x Early maturity CU LBR line |
Resistant |
|
993186-5-2 |
Early maturity CU LBR line |
Resistant |
|
E6203 |
Fully susceptible - E6203 |
Susceptible |
B. 7 genotypes, 2 plant each rep, 4 reps, 3 times, isolate US-7
|
Genotype |
Characteristics of Genotype |
Late Blight Results |
|
Legend |
Ph-2 source that is determinate |
Susceptible |
|
993154-10-3 |
CU A46-derived line - CU early maturity LBR |
Resistant |
|
Legend X NC 96LB |
Legend X LB/EB line from NC 96LB |
Light disease |
|
NC215E |
NC215E |
Susceptible |
|
NC 96LB |
LB/EB line from NC 96LB |
Resistant |
|
Legend X 993154-10 |
Legend X CU early maturity LBR |
Light disease |
|
W.V.63 |
Ph-2 source that is indeterminate |
Susceptible |
C. 11 genotypes, 8 plant each rep, 2 reps, 2 times, Isolate US-7
|
Genotype |
Characteristics of Genotype |
Late Blight Results |
|
004911-3 |
Sub-selection AVRDC CLN 2037B |
Light disease |
|
004911-5 |
Sub-selection AVRDC CLN 2037B |
Light disease |
|
004914-4 |
Sub-selection AVRDC CLN 2037E |
Light disease |
|
004914-6 |
Sub-selection AVRDC CLN 2037E |
Light disease |
|
CLN 2037 A |
AVRDC line |
Light disease |
|
CLN 2037 C |
AVRDC line |
Light disease |
|
CLN 2037 D |
AVRDC line |
Light disease |
|
CLN 2037 F |
AVRDC line |
Light disease |
|
CLN 2037 G |
AVRDC line |
Light disease |
|
CLN 2037 H |
AVRDC line |
Light disease |
|
CLN 2037 I |
AVRDC line |
Light disease |
US-11 results, Table 3 section A.
Only leaflets of the susceptible genotype E6203 showed disease symptoms and sporulation when inoculated with US-11. This is in complete agreement with prior lab tests using similar genotypes and this isolate, and in agreement with the results of the 2002 field trial using this isolate.
US-7 results, Table 2 section B.
Leaflets of the susceptible genotype NC 215E showed disease symptoms and sporulation when inoculated with US-11. The leaflets of the two Ph-2 lines, Legend and W.V. 63, showed similar high levels of disease symptoms and sporulation when inoculated with US-11.
US-7 results, Table 3 section C.
The genotypes in Section C were tested simultaneously with those in Section B of the table, but more plants per genotype were surveyed for the entries in section C since these lines had not been tested before. All of these entries were either lines from the AVRDC, or the most resistant selections from a prior test of two AVRDC lines. All of the ACRDC lines were derived using the L3708 source of late blight resistance. The lab test of all of the entries in Table 3 section C were extremely uniform, resulting in disease that was lighter than that of the susceptible lines in sections A and B of the tests, but uniformly present, in contrast to the lack of disease in the Cornell Late blight resistant lines also derived using the L3708 source of late blight resistance. The uniformity of the disease across the AVRDC lines, as well as the progeny of selections from the AVRDC lines, indicates that these lines are uniformly fixed for their reduced level of resistance. Therefore, these lines should not be used as germplasm to breeding new late blight resistance varieties.
