Busy as a Bee: July progress report.

Purpling on non-grafted Cherokee Purple at Blue Heron Farm

Every month, I write a report to my graduate committee members to track my montly progress. This season has been hectic. It is nearly 8pm, and I am still at work, ending my day just as it began, in front of the computer. Here's the monthly run-down.

Carol asked me to include some comments on my thoughts and feelings regarding the progress of my research. I enjoy gaining knowledge in plant pathology, although I still feel very overwhelmed and lacking in knowledge in this area. I am very excited for General Plant Pathology this coming semester. I enjoy being in the field much more than the lab. Thus, fruit quality analysis for my trials reflects data presented in peer-reviewed articles regarding grafted vegetables but does not go beyond that. I enjoy the opportunity to interact local growers and agriculture professionals. I enjoy working with and supervising the WSU NWREC Vegetable Horticulture staff to keep trials monitored and maintained.


I feel as if I can’t speak to where I would like to focus my research at this point in time. In the midst of the field season, I feel overwhelmed by the scope of my immediate research tasks. I get narrow-sighted, as I struggle daily to keep up with collecting quality data from my trials. I would like to collect plant samples from high tunnel-grown tomatoes throughout western Washington, but I don’t think I have the space in my schedule. It is difficult having so many trials over such a large geographical area, especially since my three crops require multiple harvests. I frequently feel as if I struggle to get everything done and organized, such that no one task gets completed in a thorough and quality manner.


1) Alan Schreiber On-farm field trials. Brian Koepke, an undergraduate student and intern through WSU stationed at Alan Schreiber’s farm, has been responsible for maintaining and monitoring the plots on a weekly basis. This includes clearly marking treatment plots, staking and trellising tomatoes, staking eggplants, pruning off rootstock re-growth, and monitoring for disease symptoms. Alan Schreiber does not prune tomatoes on his farm, so trial tomatoes have not been pruned making disease ratings difficulty.

a. Tomato.

On-farm trial at Schreiber and Sons Farm

i. Disease Severity Rating. The tomato trials were observed to have developed purpling and rolling (dissimilar to ‘physiological leaf roll’). The incidence and severity of these symptoms were monitored and photographed three times a week by Brian Koepke until July 12.

ii. Plant Assays. Two samples of affected plants were collected during site visit on July 12. Leaves were put in a moist chamber for two days and assessed for late blight (Phytophthora infestans) with negative results. A Verticillium plant assay was also prepped for both plants.

iii. ELISA Virus Assays. Leaves from these two samples were also submitted to the WSU Prosser ELISA labs to be assessed for Tomato Mosaic Virus and Beet Curly Top Virus. Both samples tested negative for Tomato Mosaic Virus but one sample tested positive for Beet Curly Top Virus. I need to do further research on Curly Top virus and host insects.

Eggplant.

Grafted eggplant trial at Schreiber and Sons Farm.

i. Disease Severity Rating. The trial was photographed and rated for disease and vigor on July 12, 2010.

ii. Verticillium Plant Assay. On June 22, twenty samples were collected from symptomatic plants in Alan’s eggplant field. These were rated by Debbie Inglis on July 15. Verticillium was not confirmed in any samples. Questionable samples were plated onto PDA. One plate yielded possible Verticillium. This was transferred onto NP10 and will be observed again in August.

Hermiston Watermelon Trials

Chlorotic watermelon leaf sample being prepared for moist chamber.

Sam Pollock On-farm Trial.

On-farm grafted watermelon trial at Sam Pollock's leased field

i. Disease Severity Rating. The trial was photographed and rated for disease and vigor on July 12, 2010. Sam Pollock has leased this field outside Hermiston. He has provided me with 2005-2010 crop history, 2009 chemical applications, and 2010 chemical applications.

ii. Stem diameter data. On July 19, Brian Koepke measured stem diameters of watermelon. Stem diameters were measured with a tape measure instead of a caliper, so they may be inaccurate. Stem diameters will be measured three times throughout growing season. If Brian’s measurements differ greatly from caliper-measured diameters, the July 19 data may have to be thrown out.

Hermiston OSU REC Trial.

Rating disease severity on grafted watermelon trial at OSU Hermiston Research Station

i. Disease Severity Rating. Trial was rated for disease symptoms, physical defects, vigor and other visible symptoms on July 12, 2010. Syngenta-grafted plants are ‘Strong Tosa’ rootstock with cv. Melody instead of cv. Crisp’n Sweet. Grafting clips were left on Syngenta grafts and our weak plants. Some clips did not pop off as plant grew, compromising vigor and stem diameter.

ii. Stem diameter data. Later in July, Brian Koepke measured stem diameters of watermelon. Stem diameters were measured with a tape measure instead of a caliper, so they may be inaccurate. Stem diameters will be measured three times throughout growing season. If Brian’s measurements differ greatly from caliper-measured diameters, data may have to be thrown out.

Mount Vernon Field Trials.

Irrigation was increased in mid July to induce Verticillium (see specific details below). . From July 20-23, irrigation was increased for a total of 1.6 inches. Following July 23, Irrigation rates were returned to an average rate of 0.5 inch every 9-10 days. Photograph documentation consists of an overview photo, a photo of each repetition, and a photo of each treatment plot in one repetition.

Eggplant.

i. Disease Severity Rating. Eggplant have been monitored and photographed weekly at 8am on Wednesday or Thursday. Physical symptoms are photographed and rated for incidence and severity.

ii. Pesticide Applications. Aphids have been observed and plants have been treated with Pyganic.

Tomato.

i. Disease Severity Rating. The trial has been monitored and photographed weekly at 8am on Wednesday or Thursday. Physical symptoms are photographed and rated for incidence and severity.

ii. Pesticide Applications. Aphids have been observed and treated with EnTrust and Pyganic. Physiological leaf roll is effecting all plants in trial with no observed treatment difference and >50% severity in most plants.

iii. Trial maintenance. Tomatoes are pruned weekly and trellised as needed.

Watermelon.

i. Disease Severity Rating. The trial has been monitored and photographed weekly at 8am on Wednesday or Thursday. Although slow-growing in the cool weather, plants remain extremely healthy. Currently, the only physical defect is slight damage to the leaf cuticle caused by windburn and cold damage on older leaves from original transplants.

PPath C3 Inoculated Field Tomato Trial.

i. Disease Severity Rating. Trial is monitored and pruned weekly at 8am on Thursday or Friday. It is being trellised as needed.

ii. Irrigation. Irrigation rate was increased to 1” per week on July 20, 2010. From June 21-July 21, 2010, trial was irrigated twice weekly with a 2 gallon watering can at an approximate rate of 0.353 inches per plant. Irrigation rate will be decreased once plants set fruit.

iii. Verticillium Soil Assay. Soil samples were collected on July 21, 2010 for a soil assay to determine inoculum levels of Verticillium in plots. 9 cores were taken from each plot at a depth of 2-4” and mixed together in plastic bags. Soil was dried for two weeks in open paper bags under a fume hood. This soil assay will be conducted in August or September.

Blue Heron Farms.

i. Disease Severity Rating. 19 plants (10 Beaufort:Cherokee Purple and 9 non-grafted Cherokee Purple) at Blue Heron Farms were monitored and photographed on July 1 and July 23, 2010 . Non-grafted CP developed purpling on leaves.

ii. Treatments. On July 22, tomatoes were treated with a foliar application of compost tea for organic disease prevention.

4) Miscellaneous.

a. Plant Pathology. Kevin showed me how to use the NWREC autoclave and how to make NP10 media (required for the Verticillium soil assay). I also had a chance to look through Lindsey DuToit’s Verticillium collection with Debbie Inglis and Babette Gunderson. I watched the eOrganic webinar on late blight (Phytophtora infestans).

b. Certified Organic Field Harvest. I have been responsible for coordinating harvest in the certified organic field.

c. NARF Field Day. I gave a short presentation to approximately 100 people at the NARF field day about the grafting project. I also attended the NARF organic working group meeting.

d. Organic Certification Inspection. I participated in the organic certification inspection on July 14.

e. Viva Farms. I coordinated with Don McMoran to include graduate students and NWREC staff in a WSU Extension Staff tour of the Viva Farms Incubator Program on July 21. I also have been assisting Don McMoran and Sarita Schaeffer with tractor work at the farm as time in my schedule permits.

f. Lycopene Analysis. Jeremy Cowan and I continue to troubleshoot the University of Florida lycopene protocol. We are getting results that vary widely from the USDA Nutrient Analysis Lab data.

g. Fruit Quality Analysis. I’ve been working with other staff to finalize the harvest and fruit quality analysis protocols for the 2010 tomato harvest. I also continue to work on the eggplant and watermelon harvest and fruit quality protocols.

h. Grafting. Patti Kreider and I have been getting practice with hole insertion technique on butternut and watermelon starts.

i. Leica Stereomicroscope. I worked with Carol to purchase a dissecting scope for the Vegetable Horticulture lab following a generous donation by Craig McConnell.



GOALS FOR AUGUST

1. Verticillium soil assay. I hope to get soil plated onto NP10 before classes start on August 23.

2. Continue to collect data on field trials. I will continue monitoring for symptom severity and incidence on field plots. My goal is to observe on-farm trials at least three times. Stem diameters in watermelon were measured again on August 3, 2010. They will be measured a third time on August 17, 2010. Stem diameters will be measured weekly for watermelon at Mount Vernon beginning on August 10. Harvest data will be collected, as possible, at on-farm trials. Fruit quality data (Texture/firmness, soluble solids/Brix, and lycopene) will be collected three times throughout harvest. Following fruit harvest, destructive samples will be taken and plants will be assayed for Verticillium.

3. Literature review. Look at other thesis examples. Make an outline for lit review. Read at least three articles each week.

Fused flowers

I chose Cherokee Purple as the scion variety in my grafting trials because it is a popular heirloom that is stingy on yield. Heirloom varieties are given to more sporadic behavior. They don't perform as consistently as the meticulously selected commercial hybrids. The standard varieties, often hybrids, are the athletes of the tomato world. They grow and flower and fruit consistently and well. The heirloom varieties are beautiful in color, mostly great in flavor, but given to some odd habits. Growth habits, specifically.

The apical meristem of the Cherokee Purple often develops an inflorescence. At the apex of this inflorescence, the stem leading to the flower (the peduncle in botany-speak) and the resulting 2-4 flowers are fused together.

This variety is also planted in a second project at the research station that is collaborating with two universities in Texas and Tennessee. We asked them whether we should remove the fruit or not, as it will likely result in unmarketable fruit. They have not been removing them, since they believe that growers wouldn't remove them in the same situation. Therefore, we have not been pruning them off.

This is a Cherokee Purple fruit developing 2 weeks after the flower photos above. This is some serious cat-facing that might result in increased susceptibility to pathogens (read: rotten and diseased fruit). Pruning off this fused flower at the apex of the main leader might be worth considering, if we choose to grow the same variety next year.

It's the end of June already!

Grafted watermelon, newly transplanted in Hermiston, OR.

It's already been a few weeks since we planted our trials out. The Pacific Northwest is experiencing an unusually rainy cool spring and early summer, so the watermelons planted out at the Mount Vernon NWREC (the research station that is my home base) are struggling. Hopefully, Cliff Mass is right, and the PNW summer will officially start after the 4th of July.

P. laying out eggplants at Schreiber and Sons Farm.

On June 7 and 8, Patti and I travelled over to the Columbia Basin to put in our grafted eggplant and watermelon trials. Our eggplant trials were planted at Schreiber and Sons farm in a field with confirmed Verticillium dahliae populations and a history of major eggplant crop loss from Verticillium.

Organizing watermelon treatment blocks in the hotel parking lot.

The following morning, we put in the watermelon trials in Hermiston, OR. Hermiston, OR is an example of irrigated crop production in the dry sagebrush country of the Columbia Basin. It has hot dry summers. Days are warm and nights are cooler allowing sugar to build up in the day but not burn off at night. Because of the day-night temperature difference, watermelons are sweeter in Hermiston than places like California and Texas. They even have a big slice of watermelon painted on the town water tower.

Hermiston also has very sandy soils. Because of this, they grow row crops interplanted between wheat. The wheat acts as a wind break. If you don't look too closely, you can drive by a seedless watermelon field and mistake it for a wheat field.

Patti transplanting grafted watermelon into field in Hermiston, OR.

Watermelon trial newly planted at Sam Pollock's field.

We will be going back over to Hermiston in a few weeks to check on the trials and monitor for Verticillium disease symptoms.

Everything is in!

Well almost... The majority is in. There are a few grafts still waiting in the healing chamber, but it's getting later in the growing season. Now I can shift my focus to pruning, monitoring, and fruit quality analysis.

We're on the final grafting stretch.

We are getting closer and closer to having all our grafting done for the field season. The on-farm eggplant and watermelon field trials will go in on June 7 and 8. The Mount Vernon field trials will go in the following week *fingers crossed*. Today we had a visit from reporters for a news article.

Patti gave a great demonstration of watermelon and tomato grafting techniques, and the reporters got to try their hand at grafting tomatoes.

Patti, grafting whiz, splice grafting Cherokee Purple tomatoes.

Patti holding Crisp'N Sweet watermelon grafts, me holding Cherokee Purple tomato grafts with healing chamber in the background.

Grafted tomato trial goes in at A. Schreiber's in Eltopia, WA

My biophysics instructors gave me the "Most likely to self-combust from stress" award. Good thing the class is almost over.

Effect of foliar application of chitosan on stomatal conductance and survival of grafted transplants.

(This project was completed for my environmental biophysics project. Tables, figures, and some symbols did not come through).

Abstract
Chitosan is a natural compound derived from chitin that activates plant defense response and stomatal closure thereby reducing transpiration. The purpose of this study was to investigate the effect of chitosan on stomatal conductance and survival of grafted Cherokee Purple and Maxifort tomato plants. All plants were self-grafted using the apical grafting technique. Chitosan solution was applied to the foliar surface of treated plants. Effect of chitosan was observed in two growth chambers held at 25C and 37% and 95% relative humidity respectively. Stomatal conductance was measured with a leaf porometer every 24 hours for eight days following grafting. Stomatal conductance was reduced in the initial hours after foliar chitosan treatment in Cherokee Purple but the effect was not sustained. Chitosan-treated Maxifort plants did not show significantly lower stomatal conductance as compared to untreated plants. Results of this study indicate no significant reduction in transpiration in grafted transplants by foliar application of chitosan. Effectiveness of chitosan foliar application in reduction of stomatal conductance and higher graft survival at lower humidity levels remains inconclusive.

Introduction
Chitosan is a natural biodegradable polymer obtained by deacetylation of chitin from crustaceans. Foliar applications of chitosan have been shown to elicit plant defense mechanisms resulting in stomatal closure and reduction in transpiration (Bittelli, Flury et al. 2001; Iriti, Picchi et al. 2009). Grafting vegetables is extensively used in Asia, Europe, Canada, and increasingly in the United States for improved plant vigor and resistance to soil-borne pathogens. During the grafting process, xylem is severed and the scion is under extreme water stress until the xylem tissue of the scion and rootstock heal together. In order to minimize water stress, efforts are made to reduce transpiration for a period of time immediately following grafting (Rivard and Louws 2006). Grafted vegetables are placed in healing chambers with optimal temperatures (20-30C), high relative humidity, between 95-100%, and complete darkness for five days(Grubinger 2007; Zhao 2010). After 5 days, light is slowly increased while humidity and temperature are slowly decreased (Hassell, Memmott et al. 2008). As chitosan is a known antitranspirant, foliar applications of chitosan to newly grafted plants may reduce water stress to scion and subsequently increase graft success. Chitosan could potentially provide a low-cost alternative to climate-controlled healing chambers for both conventional and organic growers with limited space and resources. The purpose of this study was to look at the effect of chitosan on stomatal conductance and survival in grafted plants at suboptimal and optimal humidity levels.

Materials and Methods
Chemicals
One liter of chitosan solution was prepared according to methods used by Bittelli, Flury et al. (2001) with some modification. 1 gram of chitosan (448869; Sigma-Aldrich) was dissolved at a concentration of 1 g L-1 in 100mL of 1% (w/w) lactic acid solution. MilliQ water was added until 0.1% (w/w) lactic acid solution concentration was obtained. A 0.1% lactic acid solution without chitosan was also prepared for control plants. Solutions were neutralized to pH 6.3 with sodium hydroxide (NaOH) using an automatic titrator.

Plant materials
Two varieties of tomato commonly used in grafting research, Maxifort and Cherokee Purple, were chosen for this study. Maxifort (DeRuiters, Inc.) is a commercial rootstock used in production of grafted tomatoes. Cherokee Purple (Territorial Seed Co.) is a popular heirloom tomato variety. Untreated Maxifort seed and organic Cherokee Purple seed were sown on February 22, 2010 in Sunshine Mix #1 in 72 cell trays. A data logger was used to measure temperature and relative humidity between February 22 and April 9, 2010. Average daytime temperature and relative humidity in the greenhouse were 21.6C and 27% respectively. Average nighttime temperature and relative humidity level were 12.8C and 39%.

Grafting and Chemical Application
A total of 76 plants (38 Maxifort and 38 Cherokee Purple) were grafted at 5:00am on April 9, 2010. Prior to grafting, a 10% bleach solution was used to sterilize all surfaces. Grafting was done with sterile double-edged disposable razor blades. Plants were self-grafted using the apical grafting technique, such that the angle of cut on rootstock and scion was identical and contact at graft union optimal. Grafts were held together with silicone grafting clips. Immediately following grafting, treatment plants were sprayed with chitosan solution at a rate of 10 spray-shots on both adaxial and abaxial leaf surface. Control plants were sprayed with 0.1% lactic acid solution at the same rate of application.

40 plants were placed in a growth chamber at 25C with no humidity control. Relative humidity was measured with a capacitance hygrometer every 24 hours. 36 plants were placed in a growth chamber held at 25C and 95% relative humidity (Table 1).

Light was excluded for 7 days. Stomatal conductance for each plant was measured with a steady state leaf porometer (Decagon SC-1) at 3 hours and 8 hours after grafting. Subsequent stomatal conductance measurements were collected every 24 hours over the next 7 days. As tomatoes are hypostomatous, stomatal conductance was measured only on the abaxial leaf surface. Plants were misted with 10 spray shots of water every 24 hours. On Day 8, plants were returned to the greenhouse for 2 hours with harvest crates over them to diffuse direct solar radiation. Plants were returned to greenhouse for 4 hours on Day 9. On Day 10, plants were permanently moved from the growth chamber to greenhouse.

Statistical Analysis
Statistical analysis was performed at a 95% confidence level (=0.05) using Minitab 15 (Minitab, Inc.). Two-sample t-tests were performed on Maxifort and Cherokee Purple varieties at each time interval with treatments consisting of chitosan-treated plants and untreated plants. An F-test was conducted to determine equality of variance between two samples thereby determining the appropriate t-test: pooled t-test or Welch’s t-test. Although sample size was small (n=9-10), a two-proportion comparison was conducted for percent survival of chitosan-treated plants versus untreated plants for each day of data collection. Pearson correlation coefficients were calculated for stomatal conductance and time after grafting. Correlation coefficients were also determined for standard deviation of stomatal conductance and time after grafting. Analysis was specific to variety and no comparisons were made between Cherokee Purple and Maxifort varieties.

Results
37% Relative Humidity
Although the growth chamber did not have humidity control, temperature was maintained at a constant 25C and thus the growth chamber maintained a nearly constant 37% relative humidity. Stomatal conductance decreased between 3 hours and 8 hours after grafting. Leaves were too wilted at 29 hours to accurately measure stomatal conductance using the porometer, so no further readings were taken.

At three days, all plants had wilted. At four days however, some scions began to regain turgor pressure. After eight days, there was a significant difference between percent survival of chitosan-treated and untreated Cherokee Purple plants (P0.05). At full acclimation on Day 10, chitosan-treated Cherokee Purple plants had 30% higher survival than untreated plants for both humidity levels. There was no significant difference in percent survival between chitosan-treated and untreated Maxifort plants.

95% Relative Humidity
On Day 10, there was no significant difference in survival of chitosan-treated and untreated plants healed in the 95% hr growth chamber for either Maxifort or Cherokee Purple. Maxifort plants had 90% survival on Day 10 regardless of treatment. On Day 5 and Day 6, chitosan-treated plants had significantly higher percent survival than untreated plants (P0.01). However, at completion of the experiment on Day 10, there was no significant difference between survival between Cherokee Purple treatment groups with 80% survival of chitosan-treated plants and 50% survival of untreated plants (Fig. 1).

Initially, it was hypothesized that standard deviation of stomatal conductance would increase over time, as xylem tissue of some grafts healed together while scion and rootstock of other plants remained disconnected. However, only chitosan-treated Cherokee Purple plants showed a significant correlation between standard deviation of stomatal conductance and time after grafting (P0.05).

Cherokee Purple plants that were treated with chitosan had significantly lower stomatal conductance than untreated plants 3 hours after grafting (Fig. 2). At Day 1 and Day 6 stomatal conductance for chitosan-treated plants were however significantly higher than untreated plants (P0.05).

There was no significant difference between chitosan-treated Maxifort grafts and untreated grafts at any time during the healing process (Fig. 3).

Discussion
Numerous studies have documented the antitranspirant effect of foliarly applied chitosan (Lee, Choi et al. 1999; Bittelli, Flury et al. 2001; Iriti, Picchi et al. 2009). However in this study, chitosan-treated plants did not have significantly lower stomatal conductance than untreated plants during the seven days after grafting for either tomato variety. Although stomatal conductance of chitosan-treated Cherokee Purple plants was significantly lower than untreated plants three hours after grafting, this effect was not sustained and Maxifort plants showed no significant reduction in stomatal conductance. The insignificant effect of chitosan in this study may have been due to the lack of light during the healing process, as stomata typically close in dark conditions. Also, the process of grafting possibly could have induced physiological stress responses in the plant that superseded the chitosan-induced defense response.

37% relative humidity is much lower than current research recommendations for post-graft healing, and the decrease in stomatal conductance between 3 hours and 8 hours and complete wilting at 29 hours was therefore expected (Rivard and Louws 2006; Zhao 2010). It was expected that, due to wilting, all grafts would fail. The survival of 50-60% of chitosan-treated grafts and 20-40% of untreated grafts at suboptimal humidity was unexpected. Although the greater percent survival of chitosan-treated Cherokee Purple grafts was statistically significant, the number of plants per treatment was very small. Thus, efficacy of chitosan foliar application in reduction of stomatal conductance and higher survival rates of grafts at suboptimal humidity levels remains inconclusive.

95% relative humidity is optimal for healing grafted transplants and the high percent survival of both Cherokee Purple and Maxifort support this recommended humidity level (Rivard and Louws 2006; Zhao 2010). There was no significant difference between percent survival of chitosan-treated plants and untreated plants at this level. Based on this small sample size, it appears that chitosan foliar application does not increase survival of grafted transplants healed at optimum relative humidity.

Stomatal conductance data at both humidity levels exhibited a high amount of variance. This is likely due to small sample sizes, physiological responses of scion and rootstocks to grafting, and inconsistencies of leaf porometer readings. A larger sample size would be recommended for future research. In addition, measuring stomatal conductance with the leaf porometer may have injured the graft union through slight agitation or movement of the scion away from the rootstock.

Conclusion
Results from this study do not conclusively demonstrate chitosan as an effective antitranspirant for newly grafted plants. Initial reduction in transpiration was not sustained beyond three hours after grafting for both suboptimal and optimal relative humidity levels. Chitosan-treated plants did show higher percent survival for all varieties and humidity levels. However, percent survival of chitosan-treated transplants was statistically significantly higher only in Cherokee Purple grafts at 37% relative humidity. The mixed results of this study are insufficient to conclude with confidence that chitosan is an effective foliar treatment for reducing water stress during the healing process in grafted transplants.

REFERENCES
Bittelli, M., M. Flury, et al. (2001). "Reduction of transpiration through foliar application of chitosan. ." Agricultural and Forest Meteorology 107: 167-175.

Grubinger, V. (2007, 1/07). "Grafting greenhouse tomatoes." from http://www.uvm.edu/vtvegandberry/factsheets/graftingGHtomato.html.

Hassell, R. L., F. Memmott, et al. (2008). "Grafting Methods for Watermelon Production." HortScience 43(6): 1677-1679.

Iriti, M., V. Picchi, et al. (2009). "Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure." Environmental & Experimental Botany 66: 493-500.

Lee, S., H. Choi, et al. (1999). "Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis." Plant Physiology 121: 147-152.

Rivard, C. and F. J. Louws (2006). "Grafting for disease resistance in heirloom tomatoes." North Carolina Cooperative Extension Service.

Zhao, X. (2010). Personal Communication. 4/24/2010.

Biophysics Project As Photo Essay

Top grafting old apple trees in the Tukey Orchard

During the past five months, I have been the teaching assistant for Plant Propagation (HORT 251) here at WSU. I prep labs, write, administer, and grade quizzes, grade lab reports, and entertain the students. On a wonderfully sunny day this week, we took the class out to the Tukey Orchard.

The R.B. Tukey Horticulture Orchard is just off campus and was established for research and education purposes. Surplus fruit is sold to the public and there are u-pick cherries, apples, pears, and peaches (in mild years). Many of the trees are over 30 years old. Aging trees begin to slow in productivity and decrease in yield. The orchard houses lots of varieties. Many varieties were experimental and never gained momentum while other varieties have simply fallen out of favor over the decades. The solution to an aging unfashionable orchard: top-grafting!

Top-grafting is a grafting technique used to revitalize old established orchards or replacing a variety that may be waning in popularity with a rising star (such as 'Pink Lady' or 'Honeycrisp).

In the spring, the established tree's branches are cut back close to where they arise from the trunk. It is important to top graft in the spring, as the bark 'slips' away from the inner wood due to cambium growth. The scion wood's cambium must come into contact and heal with the rootstock's cambium. Slipping the bark away from the cambium, allows you to securely place the scion cambium in contact with the rootstock cambium.

Scion on left has been slipped between bark and cambium. Scion on right has been sealed with grafters wax to prevent it from drying out.

The scion material are new shoots that are about the thickness of a pencil. The lower ends must be cut in a wedge shape to maximize exposure of the cambium and to slip securely in between the bark and inner wood of the rootstock. Cutting the scion in the perfect wedge is a lot more difficult than it initially appears and requires confidence around very sharp knives.

A vertical cut is made about 3/4" down the bark of the rootstock, the bark is gently pried away from the cambium, and the scion is pushed down in between the bark in inner part of the rootstock.

Scion is nailed in place to secure.

The base of the scion is then covered with grafters wax to prevent drying out. The scion is pruned back to 3-4 buds to promote graft healing rather than vegetative growth. All wound surfaces are painted with seal and heal to prevent water loss and disease infection. About 3-4 scions are grafted into each main branch of the rootstock.

Did I mention that I am TAing a great group of students? Well, they're great.

And they did a mighty fine job on this old fruit tree too!

Scions begin rooting on Day 5 after grafting.

It has been interesting to observe changes in graft cut and stomatal conductance each day. I believe that some of the grafts have healed together, as their stomatal conductances have increased to normal levels. If I were to do further growth chamber studies, I'd be curious to experiment with the methyl blue dye used in an experiment in Japan. Similar to the child's science experiment with celery or carnations with colored water, rootstocks are placed in methyl blue solution. When grafted onto the scion, xylem connection can be directly observed by methyl blue moving into the scion tissue. I'll add that to my long list of Maybe Someday To Do's.

Scion rooting and swelling graft cut pushing off silicone clip (Cherokee Purple self-graft)

I noticed a few days ago that the scion was swelling at the graft cut. Now most of the scions have been pushing out roots, as would be expected at high humidities. I'm hoping that these roots will naturally air prune and callus tissue is simultaneously forming and healing the scion and rootstock together.

Graduate School Lesson #518: low humidity kills newly grafted plants.

I am in Day 2 of a ten day biophysics project. I thought I would share with you some beautiful images of graft failure. In case you were curious, these are dying grafts 29 hours after grafting:

These plants have been placed in a growth chamber held at 25C. The average relative humidity in the growth chamber over the past 29 hours is a lethal 37%. Surprisingly, three of the Maxifort grafts have perked up since yesterday. We'll see if they make it through the next 24 hours.

In contrast, the grafts in the humidity-controlled growth chamber held at 25C and 95% humidity look fantastic:

The wilted Cherokee Purple in the right foreground is the only wilted plant out of 40 plants.

I will be measuring transpiration rate over the next 10 days. I'm excited to see what happens once the xylem of the rootstock and scion heal together. Will the transpiration rate increase? I hope I get some interesting results, especially since I only had access to one humidity-controlled growth chamber which meant sure death for my poor grafts placed in the 37% r.h. chamber.

Especially since I have spent a good 12 hours in the past three days taking painstaking measurements with a slow cumbersome leaf porometer. Trying to place wilted leaves in the sensor is like trying to fold origami with tissue paper and not recommended for those short of patience and quick of temper.

Field Work Begins!

It may have only taken 10 minutes, but 2010 field work started today with Carol and I staking out the eggplant field trial at A. Schreiber's farm. A. Schreiber is the largest eggplant producer in Washington State. His very diversified farm supplies 400+ with CSA shares in the Tricities area. His farm also has serious problems with Verticillium wilt which requires him to fumigate with a chloropicrin fumigant prior to each field season. As this option is not available for his organic eggplant production, he has agreed to cooperate and give us space for a grafting field trial in his eggplant field.

The field trial will consist of a RCBD (randomized complete block design) with five replications of four treatments:

1. Self-grafted 'Epic' eggplant
2. Non-grafted 'Epic' eggplant
3. 'Epic'/ Solanum aethiopicum graft
4. 'Epic'/ Maxifort graft

Drip tape will be cut from main drip tape, so soil in our plots is not fumigated. We have confirmed V. dahliae infection in this field through the Oregon State University Plant Disease Clinic. The surrounding fields will be fumigated in mid-April and planted a few weeks later. We are aiming to plant our field trial the first week of May. And so it begins...

Carol and I immediately after successfully staking out field trial and shortly before experiencing some minor vehicle problems.

Environmental Biophysics Project!

Project Proposal: Effect of foliar application of chitosan on stomatal conductance, evapotranspiration and survival of grafted transplants.

'Samba' Eggplant, 4 weeks.

Chitosan is a natural biodegradable polymer obtained by deacetylation of chitin from crustaceans (K. Ohta 1999; Marco Bittelli 2001). Foliar applications of chitosan have been shown to elicit plant defense mechanisms resulting in stomatal closure and reduction in evapotranspiration ((Marco Bittelli 2001; Marcello Iriti 2009). Grafting vegetables is extensively used in Asia, Europe, Canada, and increasingly the United States for increased plant vigor and resistance to soil-borne pathogens. During the grafting process, xylem is severed and the scion is under extreme water stress until the xylem of the scion and rootstock heal together. In order to minimize water stress, efforts are made to reduce transpiration and water stress for a period of time immediately following grafting. Grafted vegetables are placed in healing chambers with optimal temperatures (20°C) and high relative humidity (95-100%) where light is excluded for five days ((Hassell 2008). After 5 days, light is incrementally increased while humidity and temperature is slowly decreased. As chitosan reduces evapotranspiration, foliar applications of chitosan to newly grafted plants could reduce water stress to scion and subsequently increase graft success. Chitosan therefore could provide a low-cost alternative to climate-controlled healing chambers for both conventional and organic growers with limited space and resources.

METHODS

I am currently unsure what type of plant and number of plant I will use (tomato, eggplant, or mung bean), as it depends on growth stage when I have the materials for the chitosan solution. There will be a total of 80-140 grafted transplants. There will be two treatments and a control: grafted plant without chitosan foliar application, grafted plant with no treatment, non-grafted plant. 1 g/L chitosan (75-85% deacetylated) will be dissolved in a 0.1% (w/w) D/L lactic acid solution and applied to Treatment Group 1. The chitosan solution will be applied to the adaxial and abaxial sides of the leaf (a constant number of spray shots with approximately 20 mL per plant). Plants will be placed in a growth chamber at 20°C and 85% relative humidity. The abaxial stomatal conductance will be measured with a steady state leaf porometer (Decagon SC-1) every hour for a 24 hour period. Transpiration rate (E) will be calculated from stomatal vapor conductance (Gv,s) for the 24 hour period. Grafts will be held in the healing chamber for 4 days. On Day 5, they will be reintroduced to the greenhouse environment by slowly increasing light and decreasing temperature and humidity. On Day 10, total number of successful grafts for each treatment group will be counted.

REFERENCES

Hassell, R. L., Frederic Memmot, Dean G. Liere. (2008). "Grafting Methods for Watermelon Production." HortScience 43(6).

K. Ohta, A. T., N. Konishi, and T. Hosoki (1999). "Chitosan Treatment Affects Plant Growth and Flower Quality in Eustoma grandiflorum." HortScience 34(2): 233-234.

Marcello Iriti, V. P., Mara Rossoni, Stefana Gomarasca, Nicola Ludwig, Marco Gargano, and Franco Faoro (2009). "Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure." Environmental and Experimental Botany 66: 493-500.

Marco Bittelli, M. F., Gaylon S. Campbell, Everett J. Nichols (2001). "Reduction of transpiration through foliar application of chitosan." Agricultural and Forest Meteorology 107: 167-175.

On a personal tomato note...

I said that I wasn't going to buy any seeds for my garden this year. I don't know where I'll be living, I don't know if I'll have time to maintain the garden up in Marblemount, and I'll have field trials to manage. I'm famously frugal among friends and family, but seeds are my financial Achilles' heel. I couldn't help myself...

Back in December, a grower brought this tomato to my attention as 'something you gotta try'. It's the Azoychka! The name is like an old Russian man sneezing,"Ah-Ah-Azoychka!". It is indeed a Russian heirloom was brought to the United States by Kent Whealy, one of the founders of the Seed Savers Exchange, from a collecting trip in Russia. The taste is supposed to be unlike any other tomato.

Now I just need to find my Azoychkas and me a sunny place to call home for the coming season.

Cherokee Purple

We will be growing ‘Cherokee Purple’ tomato, a large purple heirloom variety, as the scion (theupper portion of the grafted plant with desired fruit) in our 2010 tomato field trial.

Bound by time and labor constraints, I had to choose only one tomato variety. It only made sense to grow a tomato variety that is popular among locals growers, so I made some phone calls to organic farmers in the Puget Sound area. Cherokee Purple, an heirloom that is “fairly stingy on yield” (Source: Territorial Seeds), won the popularity contest. There has been considerable interest in grafting heirlooms which are generally have low disease tolerance and have been selected for fruit quality rather than vigor hybrid. One of the benefits of grafting is increased productivity due to a more well-developed root system. Cary Rivard, a graduate student at North Carolina State University, has done a lot of work looking at the effect of grafting heirloom tomatoes.

I think that I, as well as many of my fellow tomato lovers out there, find myself drawn to the nostalgia surrounding heirloom tomatoes. All vegetables are the result of human selection, and therefore products of our culture. Centuries of culinary preferences have dictated the form, flavor, and function of the vegetables in our diet. Most of the cultivars available today are the result of plant breeding, a scientific process carried out at land grant universities and private seed companies, and many of the most widely available tomato cultivars focus on year-round availability, red color, and uniform fruit size.

The official definition of “heirloom” is very loose, being any open-pollinated variety that has been maintained for 50-100 years. 150 years ago in western civilization and more recently in much of the world, there were no vegetables but “heirloom vegetables”, no seeds but “heirloom seeds” saved by the grower at the end of each harvest. Many seed companies and growers now view heirlooms as vegetables that come in odd shapes and unusual colors, providing unique and high-value alternatives to the standard spherical red tomato. “Cherokee Purple” falls in the latter category, having a dark brownish-purple tinge when ripe.

I think of heirloom vegetables as the social life of plant breeding. These vegetables contain generations of vegetable growers in their speckles and lumps. They exist because of conversations between neighbors, letter correspondences between friends and family, contact between European settlers and Native Americans, and Native Americans agricultural practices deeply rooted in American soil. And most importantly, heirloom vegetables carry centuries of vegetable growers reflecting on their harvest with hopes for the next year’s harvest. It is this hope carried forth over generations that captures the imagination of many of today’s vegetable gardeners and consumers.

I digress. Back to our tomato of the hour.

The Story of the Cherokee Purple Tomato.

According to numerous sources, the Cherokee Purple tomato was given to a well-known heirloom tomato collector, Craig LeHoullier, by J.D.

Green. J.D. Green was from Tennessee and said that the tomato was over 100 years old and had been grown by Cherokee Indians in the area. Amazingly, below is a scanned copy of the original letter from J.D. Green as posted on Mr. LeHoullier's website.

Mr. LeHoullier, who maintains a collection of over 1000 heirloom tomato varieties, named the tomato variety “Cherokee Purple” and introduced it to the Seed Savers Exchange in 1991. It has since become a popular heirloom variety prized for its flavor and interesting purple hue.

Sowing seeds and lyophilizing willow bark.

Yesterday, I spent an hour in the greenhouse seeding four flats of seeds. I'm getting some starts ready for a project for Environmental Biophysics (details forthcoming - once I actually draft up the project proposal) and also to get some more material for grafting practice.

Seeds sown:

• 66 'Cherokee Purple' tomato seeds (Source: Territorial Seeds)
• 72 'Pumpkin-On-A-Stick' (Solanum aethiopicum) seeds (Source: Abundant Life Seeds)
• 144 Mung Bean

"Mung Bean?!"

Well, what can I say. I got side-tracked. I am raising the mung beans for a willow cofactor experiment that my plant propagation professor and I are playing around with.

I am currently a teaching assistant for Plant Propagation (HORT 251). We have been doing a series of experiments on auxin-induced rooting. Auxin is a plant hormone that induces rooting in plants. The hormone is produced in the terminal and axillary buds of a plant. Auxin moves from the shoots to the roots but not from the roots to the shoots (transfer occurs in one direction). However, auxin is not solely responsible for root induction. Rooting also requires cofactors, other chemical compounds, for auxin-induced rooting to occur.

It is commonly known that willow species (Salix sp.) root extremely easily. You can plant them vertical, horizontal, upside-down; you can weave them into sculptures or bundle them into

willow-cutting burritos, and, given moisture, they will root. One common gardening trick is to soak willow twigs in water and then use the resulting willow-infusion to water your plants, thereby increasing root development and overall plant vigor.

It is important to note that willow's rooting is not necessarily due to elevated auxin production in willows but rather in the willow cofactors. Willows contain high amounts of salicylic acid, the main ingredient in aspirin. Adding aspirin to flower water is reputed to make flowers stay fresh longer. Therefore, it could be that salicylic acid may be the rooting cofactor.

Most rooting hormone products such as Hormex consist of synthetic auxin (usually IBA or 1 H-indole-3-butyric acid) mixed with talc or other inert ingredients. The cut end of the stem is then dipped into the auxin to increase rootings (some auxin will be produced in the buds).

Dr. Kumar and I wanted to look at whether a compound could be made from willowbark and talc that would similarly induce rooting in cuttings. So, I went out and harvested some young willow branches from a large tree on campus.

We then soaked these willow branches until the buds began to swell, as we thought bud development would likely coincide with increased synthesis of auxin and cofactors. Once the buds had begun to swell, the bark was peeled from the branches and submersed in liquid nitrogen. Liquid nitrogen is -196C (-321F) and quickly freezes plant material causing plant cell walls to rupture. The bark was then put then put in a lyophilizer for 24 hours. Lyophilization removes moisture from the material by exposing the material to very low temperatures in a high vacuum.

The freeze-dried willow bark can then be ground into a powder using a coffee grinder.

Once the mung beans are big enough, we will use them to test the effect of willow-bark at different concentrations on rooting. And we will see where we can go from there.

Results from the "To Soak or Not To Soak" Eggplant Experiment

After 2 weeks, I had a 68% germination rate for the unsoaked eggplant seeds and a 92% germination rate for the soaked eggplants. Based on this small trial, I am inclined to believe that soaking the seeds for 24 hours improves germination. I will be seeding more eggplant this afternoon, so I began soaking my seeds last night.

The eggplants have become infested with thrips. The greenhouse folks have been treating them (see yellow sticky paper in photo below) but they say its a constant battle unless you want to resort to continual pesticide application.