BIOAg 2011 Progress Report

Transcription

1 IOAg 2011 Progress Report TITLE: PHYTONUTRIENTS AND GENOMICS OF ORGANIC TOMATOES: SOIL FERTILITY AND/OR PLANT DEFENSE PRINCIPAL INVESTIGATORS: Preston Andrews and Amit Dhingra, Department of Horticulture and Landscape Architecture, Washington State University, Pullman, WA KEY WORDS Antioxidants, aphid, ascorbic acid, conventional, fertilizer, fruit, genomics, herbivory, lycopene, mineral elements, nitrogen, organic, pests, phenolics, phytochemicals, phytonutrients, plant defense, soil fertility ASTRACT In previous studies, we showed that apples, strawberries, and tomatoes (IOAg funded) grown with organic nitrogen sources had higher phytonutrient contents than those fertilized with readily available sources of nitrogen. While soil-based carbon/nitrogen may be the principal contributor to these differences, it is possible that increased pest herbivory in organic crops stimulates defenses that elevate phytonutrients. In this study we are testing the relative contribution of management and herbivory on phytonutrients in tomatoes and gene expression differences among these treatments. At this stage in the project, we have found that organically fertilized tomato plants, which had aphids introduced to them at the beginning of the experiment, had negligible aphids on them at the end of the experiment compared to conventionally fertilized plants. These results may be due to the poorer digestability of the leaves of the organic plants, which had lower nitrogen concentrations than the leaves of the conventionally fertilized plants. The organic leaves also had higher concentrations of potassium, calcium, magnesium, and sulfur than the conventional leaves, but lower phosphorus concentrations. The organic ripe fruit had higher phenolics and lycopene concentrations and antioxidant activity than the conventional fruit, rendering the fruit potentially more nutritious. We are continuing to analyze fruit for ascorbic acid and will soon be performing the genomic analysis. PROJECT DESCRIPTION In the winter of we grew Oregon Spring, a determinate tomato cultivar, in pots in a greenhouse in either a conventional (SUNGRO Sunshine Mix #1, LC1) or organic (LC1:Whitney Farms Organic Planting compost:topsoil (75:20:5) soil media and fertilized with either conventional (Peters Professional Peat-Lite Special and superphosphate) or organic [Westbridge iolink Organic All-Purpose 5-5-5, Micronutrient (2% Fe, 2% Mn, 3% Zn), and Cal Plus (6% Ca)] liquid fertilizers. Half of each of these treatments was grown in exclusions cages in the presence (+aphid) or absence ( aphid) of introduced green peach aphids, which were reared in the Entomology greenhouse. Thus, there were a total of four treatments (CON/+aphid, CON/ aphid, ORG/+aphid, ORG/ aphid) grown in a randomized complete sixblock experimental design under 14-hour daylength with supplemental lighting (1000 W metal halide) at o C day/ o C night temperatures. The greenhouse experiment was continued until each tomato plant had produced at least six red ripe fruits, however, most plants produced more than 10 ripe fruits. Each red ripe fruit was harvested sequentially as it reached maturity, it was weighed, its soluble solids were measured with a refractometer, and a sample of pericarp tissue from the fruit s equator was chopped, frozen in liquid nitrogen, and stored in a 80 o C freezer. After the minimum number of red ripe fruits were harvested from each plant, the remaining unripe fruits were harvested, their stage of development recorded (i.e. immature green, breaker, turning, pink and light red), their individual weights measured, and then pericarp tissue was chopped, frozen, and stored as for the ripe fruit. After all fruits were removed, the vines were destructively harvested and the fresh and dry weights of leaves, stems, and roots from each plant were determined. Total fruit yields and yield efficiency (fruit yield per unit vine weight) were calculated. Samples of leaf tissue was frozen in liquid nitrogen and stored for mineral analysis and biochemical assays. The potting media was collected at the conclusion of the experiment for future mineral analysis. Aphid counts of leaf 1

2 sub-samples from each plant were used to estimate the total aphid infestation on each plant. Since some of the caged plants had become infested with flying white flies after the green peach aphids were introduced and pest control could no longer be used, sticky traps were placed within each cage in order to estimate the relative amount of white fly infestation. White fly counts will be used as covariates in the statistical analysis. Ripe pericarp tissue from individual fruits of different weights was analyzed for total phenolics, lycopene, and total, hydrophilic and lipophilic Trolox Equivalent Antioxidant Capacity (TEAC). Leaf tissue was pooled across pairs greenhouse blocks and sent to the University for Idaho Analytical Sciences Laboratory for mineral analysis. Ripe pericarp tissue was pooled across pairs of greenhouse blocks for isolation of total RNA. Pooled samples were pulverized using a freezer mill. Total RNA was isolated using custom modifications of the Qiagen RNEasy plant RNA isolation kit. Quality control analysis was performed on each RNA sample followed by extensive molecular biology manipulations to prepare sequencing-ready cdna template. riefly, each RNA sample was converted to cdna, sheared using a nebulizer, polished using T4 DNA polymerase, ligated to barcoded sequencing oligonucleotides and checked on a bioanalyzer for quality. The samples are currently in the wait list for generating quantitative transcriptome data from each sample. SUMMARY OF PROJECT AND PROGRESS In previous studies, we showed that apples, strawberries, and tomatoes grown with organic nitrogen fertilizer sources had higher phytochemical contents than those fertilized with readily available sources of nitrogen. It is also possible that increased pest herbivory in the field stimulates plant defenses that elevate these phytochemicals in organic crops. In this study we are testing the relative contributions of soil fertility management and herbivory to phytochemical accumulation. To accomplish this we conducted a greenhouse study of tomato plants grown in exclusion cages, fertilized with either organic or readily available nitrogen sources, and exposed or not to herbivory by aphids. We harvested individual red ripe and immature green fruit and vegetative tissues of plants the plants, and are measuring the phytochemical concentrations/contents over a range of fruit sizes. The most significant findings to date are that the yields of green immature fruit and total vine weights were slightly greater for the conventionally fertilized tomato plants than for the organic fertilized. Nevertheless, these findings in favor of the conventional treatment are overshadowed reduced aphid infestation of the organically fertilized plants, as well as the higher concentrations of phenolics and lyocpene, antioxidant capacity, and soluble solids (sugars) content of the organically grown fruit. Infestation of the vines by aphids also resulted in higher concentrations phenolics and lycopene than when aphids were excluded. Leaf concentrations of potassium, calcium, magnesium, and sulfur were also higher in the organically grown vines, but nitrogen and phosphorus concentrations were higher in the leaves of the conventionally grown vines. We have also isolated RNA from the fruit, prepared sequencing-ready cdna libraries and await quantitative transcriptome sequence data. OUTPUTS Work Completed: As previously reported, total fruit yields were higher for the conventional (CON) fertility treatment than the organic (ORG) treatment, but this difference was due to yields of immature green fruits that remained at the end of the greenhouse experiment and not to red ripe fruits harvested during the experiment. There were no differences in fruit yields due to the aphid treatment. Vine fresh weights were greater for the CON treatment than the ORG treatment, with no differences due to aphid treatment. The yield efficiency of immature fruit was notably different (P=0.07) between the CON (0.61 g fruit yield/g vine weight) and ORG (0.49 g fruit yield/g vine weight) treatments, with no effect of aphid treatment. If the experiment had continued until all immature fruit had ripened, the yields and yield efficiency for red ripe fruits in the CON treatment would likely have exceeded those in the ORG treatment. There were no statistical differences in the number of immature green ( ), red ripe ( ), or total fruit ( ) among treatments. Despite there being no 2

3 statistical differences in the fruit yields, yield efficiency, or fruit numbers of red ripe fruit, the average weight of red ripe fruit was significantly greater (P<0.001) for the CON treatment (121 g) than the ORG treatment (99 g), with no effect of the aphid treatments (106 g +aphid, 113 g aphid). There were no interactions between soil fertility and aphid treatments for fruit or vine growth measurements. One of the most remarkable findings is that the ORG/+aphid treatment had significantly fewer aphids than the CON/+aphid treatment, and statistically identical to the ORG/ aphid and CON/ aphid treatments (Fig. 1). This may be due to increased phytochemical defenses (e.g. phenolics) in the organically fertilized plants. Indeed, total phenolics were higher in the ORG fruit than in the CON fruit (Table 1). Unexpectedly, the presence of aphids on the plants resulted in significantly lower total fruit phenolics than fruit from plants where aphids were not present. Similar responses were seen for fruit lycopene concentrations (Table 1). Total (hydrophilic plus lipophilic fractions) Trolox Equivlant Antioxidant Capacity (TEAC) was higher for the ORG fertility treatment and for the CON fertility treatment (Fig. 2). There was no effect of aphid treatment on total TEAC. There were significant effects on fruit soluble solids content ( o brix) for the soil fertility treatments (P=0.009) and fruit size (P=0.01), but there was no effect from the aphid treatment. The ORG fruit had 1% higher fruit soluble solids content than the CON fruit (6.03% vs. 5.04%). There were also higher levels of most mineral elements in the leaves of the ORG fertility treatment than in leaves of the CON fertility treatment, except nitrogen and phosphorus (Table 2). 5 A CON/+aphid CON/ aphid ORG/+aphid ORG/ aphid Treatments Figure 1. Green peach aphid numbers/g leaf of Oregon Spring tomatoes grown in a 2x2 factorial experiment with conventional (CON) or organic (ORG) fertility and infested with green peach aphids (+aphid) or not infested ( aphid). P=0.04, P=0.003, and P=0.04 for the soil fertility and aphid main effects and interaction, respectively. Table 1. Total phenolic and lycopene concentrations of Oregon Spring red ripe tomato pericarp tissue in a 2x2 factorial experiment with conventional (CON) or organic (ORG) fertility and infested with green peach aphids (+aphid) or not infested ( aphid). There was no significant interaction between fertility and aphid treatments. 3

4 Compounds Fertility treatments Aphid treatments CON ORG P value + aphids aphids P value Phenolics (gallic acid/g FW) < Lycopene ( g/g FW) A CON ORG Fertility treatments Figure 2. Total Trolox Equivalent Antioxidant Capacity (TEAC) of Oregon Spring red ripe tomato pericapr grown in a 2x2 factorial experiment with conventional (CON) or organic (ORG) fertility and infested with green peach aphids (+aphid) or not infested ( aphid). P=0.007 for the soil fertility treatment and P=0.56 for aphid treatment. Table 2. Leaf mineral element CON ORG P value Nitrogen (%DW) Phosphorus (ppm) 14,167 11, Potassium (ppm) 58,500 64, Calcium (ppm) 39,500 47, Magnesium (ppm) 8,467 9, Sulfur (ppm) 16,167 36,000 <

5 Our progress on this project has resulted in the completion of Objectives 1 and 2, with Objective 3 mostly complete, except for ascorbic acid analysis, and Objective 4 underway: 1. Grow a short-season tomato cultivar in insect containment cages in a greenhouse environment using a factorial experimental design (Andrews, year 1), with the following treatments: a. Soil fertility supplied with either soluble, ionic fertilizers or organic amendments and fertilizers b. Caged plants exposed or not to leaf-feeding pests (i.e. aphids). 2. Determine crop yield, plant weight, fruit quality, and extent of pest damage at the conclusion of the greenhouse experiment (Andrews, year 1). 3. Analyze phenolic, ascorbic acid (vitamin C), and lycopene contents and antioxidant activities of tomato fruit and leaves at different stages of crop maturity (Andrews, year 1). 4. In year 2, conduct functional genomic and statistical analysis (Dhingra) of leaves and fruit of select samples, in order to identify key gene activities and markers to examine the relational associations between the plant and the imposed environmental stimuli of soil fertility and insect herbivory. Publications, Handouts, Other Text & Web Products: No publications, handouts, or Web products have been produced yet during the first year of this project. Outreach & Education Activities: 1. L. Gustafson (M.S. student) presented his proposal seminar for this project on Feb. 24, 2011 in Hort 510 (Horticulture Seminar). 2. P. Andrews was an invited speaker at the International Fruit Tree Association s annual conference in Pasco, WA on March 2, 2011, where he presented Linking Fruit and Soil Quality (Health). 3. P. Andrews was a guest lecturer in SoilS 101 (Organic Gardening and Farming) on March 24, 2011, where he presented The nutritional benefits of organic food. Is it healthier? 4. L. Gustafson (presenting author), P. Andrews, and A. Dhingra presented a poster, Phytonutrients and Genomics of Organic Tomatoes: Soil Fertility and/or Plant Defense, on this project at the Tilth Producers of Washington annual conference in Yakima, WA on Nov , IMPACTS Short-Term: The tomato greenhouse experiment was completed, as well as most of the phytochemical analyses. Following the genomic and statistical analysis of our data, we will prepare a manuscript(s), which should lead to future federal grant applications following publication. Intermediate-Term: We expect that the knowledge gained from this and subsequent projects on the phytonutrient concentrations and genomics of fruit crops will permit us to determine the genetic basis for nutritional quality and to develop more effective farming methods in order to produce more phytonutrient dense crops. Long-Term: Enhanced sustainability of agriculture and improved public health. ADDITIONAL FUNDING APPLIED FOR / SECURED Additional funding has not been applied for yet, as we are waiting for our genomics results in order to make our grant application more competitive. GRADUATE STUDENTS FUNDED Luke Gustafson, M.S. Horticulture, began in August 16, 2010 and plans to graduate in Spring 2012 semester. RECOMMENDATIONS FOR FUTURE RESEARCH The next step in this research program would be to conduct field experiments with additional crop species, including leafy green vegetables and perennial fruit crops, in order to demonstrate responses under actual farming conditions and for a broader range of species. More detailed genomic and phytochemical analyses, possibly using metabolomics, would be conducted in future studies. 5