Develop eco-friendly postharvest practices to extend the shelf life of small fruits-Use of volatiles

DEVELOP ECO-FRIENDLY POSTHARVEST PRACTICES TO EXTEND THE SHELF LIFE OF SMALL FRUITS-USE OF VOLATILES

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

OTHER GRANTS

Reporting Frequency

Annual

Accession No.

1018172

Grant No.

2019-38821-29038

Cumulative Award Amt.

$498,637.00

Proposal No.

2018-04897

Multistate No.

(N/A)

Project Start Date

Dec 15, 2018

Project End Date

Dec 14, 2023

Grant Year

2019

Program Code

[EQ]- Research Project

Recipient Organization
VIRGINIA STATE UNIVERSITY
(N/A)
PETERSBURG,VA 23803

Performing Department
Agricultural Research Station

Non Technical Summary
Significant postharvest losses (up to 50%) of fresh produce is reported every year due to physiological degradation and fungal attack associated with their high nutrient content. Generally, cold storage and fungicides are used as a standard method to control postharvest disorders and diseases. However, due to growing public concern about residual chemicals in fruits and vegetables, fungicide alternatives need to be developed. We are proposing this collaborative research project to build research, teaching and extension capacity in the newly established Postharvest Research Program at Virginia State University. The goal of the research program is to extend shelf life of small fruits by innovative and sustainable postharvest practices, however, in this proposal we will focus on strawberries and once the capacity is developed, we will expand our focus to other small fruits.Plants produce volatiles as a natural self-defense system. We are proposing to use plant volatiles right after harvest to extend fruit shelf life. The objectives are: 1) to determine the effects of volatile treatments on shelf life, postharvest properties, cell wall structure and ethylene production during storage of strawberries, 2) to identify molecular changes (gene expression, quantitative proteomics and integrated modules) in strawberries upon volatile treatments, and 3) to build the capacity in research, teaching and extension (develop human capital, develop an innovative postharvest practice and disseminate the results). Outcomes of the project will strengthen the Postharvest Research Program at Virginia State University (VSU), and expand the research focus at Delaware State University (DSU), and Tennessee State University (TSU).

Animal Health Component

60%

Research Effort Categories

Basic

40%

Applied

60%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
503	1199	1020	100%

Knowledge Area
503 - Quality Maintenance in Storing and Marketing Food Products;

Subject Of Investigation
1199 - Deciduous and small fruits, general/other;

Field Of Science
1020 - Physiology;

Keywords

fungal disease

fungicide alternatives

Goals / Objectives
Objective 1: to determine effects of volatile treatments on a) postharvest properties (shelf life & fruit quality) and the level of microbiological counts on fruit surface, and b) cell wall structure and ethylene production during two weeks of storage (Drs. Taghavi and Kim, VSU).Objective 2: to identify molecular changes in strawberries upon volatile treatments through, a) gene expression analysis (Dr. Melmaiee DSU), b) quantitative proteomics analysis (Dr. Zhou TSU, Dr. Thannhauser, USDA-ARS) and c) to develop molecular modules for volatile functions in postharvest of strawberries (Drs. Zhou, Melmaiee & Taghavi).Objective 3: build capacity in research, teaching and extension (all five key personnel) by a) developing human capital, b) developing sustainable and innovative postharvest practice(s) and disseminating the results to the interested public members and research community.

Project Methods
Objective 1a: Effect of five volatiles on shelf life, fruit quality and microbiological loadIn Year 1, two strawberry varieties ('Allstar' and 'Flavorfest') will be planted in open field at Randolph Farm under plasticulture production system (2 m center rows, 30 cm apart, and 20 cm on the row) and managed based on the recommendations provided by the Mid-Atlantic Berry Guide for Commercial Growers (Demchak & Kirsten 2010). Harvested fruits (2/3rd of the fruit turns red) will be packaged in clamshells containing five different volatiles (added to absorbent pad) and control plot with water (replacing the volatile).The harvested fruits will be stored at 4 °C for 2 weeks and fruit quality parameters will be assessed right after harvest and every week for two weeks, using standard protocols (Shafiee et al. 2010). Fruit quality parameters are: weight loss, fruit decay, fruit firmness, soluble solid content, titratable acidity, total anthocyanin, vitamin C, total phenolic content and color.Microbiological loads (aerobic mesophiles, yeast and mold count before and after storage) on fruits will be assessed by counting total aerobic mesophile (TAM), yeast and mold (YM) counts on the fruits before and after storage at 4°C, following the described procedure (Kim et al. 2015 & 2017). Based on the data, the top two volatiles in extending the shelf life and the most volatile-sensitive strawberry variety will be selected.Objective 1b: Effect of two selected volatiles on postharvest properties, cell wall structure and ethylene productionIn project Year 2, the field experiment will be repeated using the selected strawberry variety. The harvested strawberries will be packaged in clamshells containing volatiles added to absorbent pads. The volatile treatments include each individual volatile (the two selected in the first year) as well as a mixture of the two volatiles (at minimum inhibitory concentration), fungicide (Switch, 62.5WG, Syngenta, positive control), and water (negative control). Immediately after harvest, packaged fruits will be assessed for fruit physio-chemical properties, microbiota assessments and gene expression and proteomics studies. Duplicate experiments will be carried out at Year 3 to determine year to year variation of these data.Objective 2a. Gene expression analysis by quantitative real-time PCR (Dr. Melmaiee, DSU)Strawberry fruits received from VSU will be used to extract total RNA using TRIzol reagent (Invitrogen, Carlsbad, CA). Genomic DNA will be digested enzymatically with rDNAse (Life Technologies, Grand Island, NY). Complete removal of DNA will be confirmed using polymerase chain reaction (PCR) with known primers that can amplify intronic regions. The DNA-free RNA samples will be used to synthesize cDNA with ProtoScript M-MuLV First Strand cDNA synthesis kit (New England BioLabs, Beverly, MA).We have selected a list of genes involved in biological processes influencing the firmness of strawberries during ripening (Table 1). The qPCR analysis will be carried out following the protocol developed in Dr. Melmaiees' lab (Kalavacharla et al. 2011, Melmaiee et al. 2015). Briefly, concentration of cDNA will be equalized for all the samples and qRT-PCR analysis will be carried out.Objective 2b: Quantitative proteomics analysis (Dr. Zhou TSU, Dr. Thannhauser, ARS)The soluble proteins and cell wall proteins will be extracted separately following a protocol (Thannhauser et al. 2013) with modifications for the strawberry fruits. Strawberry samples will be ground into a powder in liquid nitrogen, homogenized in extraction buffer and protease inhibitor cocktail from Sigma), followed by centrifugation for 30 min at 15,000 × g. The supernatant contains total soluble proteins. Pellets containing cell wall materials will be washed sequentially in buffers with increased sucrose concentration with centrifugation between washes (Feiz et al. 2006 and Verdonk et al. 2012). Cell wall enriched proteins will be extracted using a buffer according to Printz et al. (2015). Both cell wall and soluble proteins will be concentrated by Amicon Ultra-15 10K centrifugal filter device (Millipore), and dissolved in TMT labeling buffer. Protein concentration will be measured using a Bradford assay (Ye et al. 2016).The tagged analytical cohort pool will be fractionated by high pH reversed-phase liquid chromatography (RPLC) followed by low pH nano-scale RPLC (Wang et al. 2011). The MS/MS spectra of labeled peptides will be generated on an Orbitrap Fusion Trybrid mass spectrometer.Objective 2c: Developing an integrated module for volatile function in strawberry postharvestTranscript and protein changes of the selected genes will be compared to determine the corroboration of gene expression at these two levels. The biological functional pathways for these genes and proteins will be predicted using STRING Database (https://string-db.org/). We will use protein/gene names to search database from Arabidopsis thaliana which has the best annotation among all plant species. The annotated biological/molecular functions of the genes/proteins will be associated with the chemical/physiological properties of strawberry (measured in Objective 1) to develop the action mode of the volatile treatments.Objective 3: Build capacity by a) developing human capital, b) developing postharvest practices and disseminating the resultsObjective 3a: Developing human capital (Student Leadership Development):All five key personnel will be involved in the training of the human capital. At VSU, a postdoc will be hired and trained to carry out thee research/extension activities and manage undergraduate student. The postdoc will be involved in all areas of the research project. He/she will travel to DSU and TSU in year 2 &3 to receive hands-on training, on the gene expression and proteomics. At the beginning of each year an undergraduate student will be hired and trained to carry out the research project. We train the student to work as a team and provide hands-on training, critical thinking, problem solving, and communication skills. At each DSU and TSU, two undergraduate students (one/year) and one postdoc (part-time, one/university) will be trained to build the capacity for research and education and carry out the project.Objective 3b: Developing sustainable postharvest practice(s) and disseminating the results:The results of the year 1 and 2 will be used to identify the most two effective volatiles in extending the shelf life of strawberries during storage. Year 3 data will be used to confirm the results and identify the interactions between the two volatiles. At least three and up to six peer-review publications will be developed (1-2 per university).Drs. Taghavi (PI) and Rafie (Co-PI) will develop a coordination plan to develop two extension fact-sheets to provide research-based information to berry growers and in particular to strawberry growers. For example, at least one and up to two postharvest practices will be developed as how to effectively and efficiently apply the selected volatiles and what combination to choose. These information will be added to the VSU Small Fruits and Vegetable (https://www.ext.vsu.edu/horticulture/) and eXtension websites. Dr. Rafie (Co-PI), will work with eXtension to organize a community of practices and link the project fact sheets. eXtension is a natural outlet for the project and is used by extension agents, students and academics which will help reach a broader demographic of audience.Each PI/Co-PI will attend at least one regional or national symposium or stakeholders meetings (i.e. North American Strawberry Growers Association and American Society for Horticultural Science annual meetings). PI will also attend the International Strawberry Symposium (ISHS in Italy).

Progress 12/15/18 to 12/14/23

Outputs
Target Audience:Target audience: Training in the lab and research field: New Virginia Cooperation Extension (VCE) agents visited the lab. On 10/17/2019. A short presentation was given to about 20 agents about the activities and research projects in the lab. Two graduate students were trained for two sessions on the research project running in the lab. The following personnel were trained in the field and the lab for (from three months to 4 years) on this project on the new and cutting-edge technologies in Postharvest Practices. They were trained to review research literature, develop the experimental design, conduct lab analysis, collect, summarize, analyze data, and prepare paper drafts. Eighteen undergraduate students (Michelle Opoku, Carissa James, Monae Bell, Shannon Lee, Efam Taylor, Krystle Jordan, John Chilaka, Ariel Coleman, Jacquelin Bullock, Michael Bushema, Burgundy Brown, Ceniya Ferguson, Joanetta King, Franchesca Synsmir, Kendyl Barnwell, Tyliah Swann, Kennedy Brandon, Milan Martin) One intern (Tariq Garrison) One research Rotation (Latia Jackson) Three graduate Students (Mzuna Aldossari, Kajol Pradhan, Ghadeer Bahamdan). One of the graduate students conducted her master's thesis jointly at VSU and Tennessee State University on this project and received training. Four technicians (Margaret Minja, Hiral Patel, Lolo Akandae, Ismail Mokhasel) One postdoc (Dominiq Galam) The intern was trained through collaboration with the Engineering Department at VSU and industry (CCAM) to develop algorithms and machine learning techniques to use hyperspectral image data for early identification of strawberry diseases. The postdoc was sent for extra training to Delaware State University for three weeks and learned techniques on transcriptome analysis (as suggested in the proposal). Four of the undergraduate students presented posters at ARD symposiums (Association for Research Directors of HBCUs), and many of them presented their research at VSU Undergraduate Symposiums. Formal classroom instructions, Laboratory instruction training Two laboratory sessions (4/11 and 4/18/2019) training were given to ten BIOL 418 (Plant physiology) students on using essential oils to improve postharvest fruit quality of strawberries. Two sessions of formal classroom training were held (on 10/22/2018 and 9/17/2019) for students of AGRI 140 (Intro to Sustainable Ag & Society), and about 40 students participated in each session. One formal class (11/6/2019) session was given to students of BIOL130, and one formal class (11/25/2019) session to students of the AGRI140 class. Each class had about 20 students on average. Two laboratory (11/1/2019 and 11/6/2019) training sessions were given to eleven students of the course BIOL 310 (Plant Morphology) One formal class for graduate students (Plant Morphology-BIOL 521) was taught for the fall 2021-2022 term. Six master students were trained on the new and cutting-edge knowledge in Plant Morphogenesis. Field tour During August each year (2018-2023), the AgFest Field Day was held at the Randolph Research Farm, and about 1000 visitors were provided with information on the research project and results. Extension through presentations Presentation at ISHS Vth International Symposium on Postharvest Pathology, liège, Belgium, March 18-25, 2019, titled: "Strawberry fruit decay is affected by plant volatiles." Presentation at Association of Research Directors (ARD) Research Symposium 2019, Jacksonville, FL, March 30-April 3, 2019, titled: Plant Volatiles: Can They Extend the Shelf Life and Reduce Fruit Decay of Strawberries (Fragaria × ananassa)? Presentation at the American Society for Horticultural Science (ASHS) annual general meeting on "Response of Strawberries to Postharvest Application of Plant Volatiles." Three presentations were made at VSU Field Day for about 100 attendees, including growers and other stakeholders (titles: 'Low tillage strawberry raised bed system', 'Application of clover cover crop to improve strawberry yield and quality', High tunnel soilless strawberry system'). One presentation on increasing collaboration among universities was made at the HBCU/MSI Research Summit at Virginia Tech University; about 50 people attended the event. One oral presentation was made virtually at the 9th International Strawberry Symposium in Italy. Many from all over the world attended the meeting. A workshop was organized on 'Immune-Improving Medicinal Plan Supplements and Challenges in the Time of a Pandemic' at the American Society for Horticultural Science (ASHS) annual general meeting. About 20 people attended in person, and many virtually. One presentation was made at VSU Field Day for about 200 attendees, including growers and other stakeholders (title: High tunnel soilless strawberry system). One oral presentation was made virtually at the 9th International Strawberry Symposium in Italy. Many from all over the world attended the meeting. A workshop was organized on 'Application and production of medicinal plants for human, plant and animal health' at the American Society for Horticultural Science (ASHS) annual general meeting. About 20 people attended in person, and many virtually. About 40 high school students were trained in the lab during AgDicovery 2023. One presentation was made at VSU Field Day for about 200 attendees, including growers and other stakeholders (title: High tunnel soilless strawberry system). One oral presentation was made at the Asian Horticultural Congress in Japan. Many from all over the world attended the meeting. A workshop was organized on 'Application and production of medicinal plants for human, plant and animal health' at the American Society for Horticultural Science (ASHS) annual general meeting. About 20 people attended in person, and many virtually. Changes/Problems: The start date of the project was December, however, the first activity is planting strawberries in the field which should have be done in September. Therefore, in the first year of the experiment, we did not have strawberry varieties that we planned in the proposal and we used other strawberry varieties and developed the protocols. Therefore, the project was delayed for a year. The hiring process of the post doc and technicians is very lengthy at Virginia State University. Three postdocs were shortlisted (one in each year) and their application were sent to HR. We were waiting for the hiring process for 6-8 months. Two of them found other positions and were not available anymore. One postdoc was hired in the third year of the project. His OPT visa terminated in a year and he had to leave the US due to not being authorized to work. We had to start the hiring process all over again and were not able to hire another postdoc due to lengthy process at VSU and very tight job market. Instead we hired a technician. The technician have found other positions outside of VSU and separated from this project During the whole project we could only hire a postdoc for a year and one technician for one year. The strawberry plants were purchased in the third year of the project from a reputable nursery were infected with the anthracnose disease. We lost four cultivars out of five planted. Due to the Covid 19, the campus shut down on March 30, 2020 for three months. Strawberries were ready for harvest on April 1st. Therefore, we lost the harvesting window of strawberries. After three months, we were not still allowed to work full time and were working in half capacity. Even so, the campus shut down twice for 14 days due to reports of department members that had Covid 19 or were in contact with someone with Covid 19. Due to the Covid 19, students were not allowed to work in the labs or the research fields. All activities were slower than the normal situation. Therefore, due to the pandemic and lockdown, we were not able to harvest any crop from our research plot in the second year of the project. This delayed the project for the another year. In the third year of the project we were not able to acquire one of the promised strawberry varieties from nurseries; therefore, the project included only four out of five varieties. In the forth year, we were not able to acquire two of the promised strawberry varieties from nurseries; therefore, the project included only three out of five varieties. In the fourth year, due to the Covid 19, students had to quarantine when they were sick and this delayed the project. All activities were slower than the normal situation due to the pandemic lingering effects. The financial report was not given to the PI at the end of fifth year even though several request were made for the report. Therefore, a request for extension could not be filed. What opportunities for training and professional development has the project provided?Three technicians, eighteen undergraduate, three graduate students, and one postdoc are receiving training. The postdoc was also sent to DSU for supplemental training as suggested in the proposal. We have developed several laboratory protocols to assess the fruit and nutritional quality at our lab. We have developed protocols to measure sugar, titratable acidity, vitamin C, fungal contamination and ethylene. We also have developed methods to assess total anthocyanin, total antioxidant and total polyphenols in strawberries, culture and co-culture of beneficial bacteria and postharvest fungi. Technicians and students were involved in almost all parts of these efforts and received training. How have the results been disseminated to communities of interest?The results were disseminated through formal classroom, Laboratory instructions, and presentations at the local, national, and international symposiums. Results were also disseminated to the 1000 growers, extension agents and many other stakeholders who attended the VSU field day during 2018-2023. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Five essential oils were screened for their efficacy against strawberry pathogens by placing 30 ul of them on a cotton ball inside the containers. Although most of them were effective in controlling the disease compared to control (no treatment), however two of them had less after-taste flavor. Also, efficacy of essential oils to reduce microbial load on fruit surface was tested. The first year results were not significant and more testing is required to make any conclusion. Fruit color is an important factor in assessing fruit quality. Six methods have been developed and compared to assess anthocyanins in strawberry fruits. The extracted pigments were sent to a lab for HPLC analysis and identification of the pigment. Four manuscripts were published as a result. Objective 2: Due to the pandemic and lockdown at the campus, no strawberry fruit could be harvested form the experimental plot in 2020. Therefore, these activities were postphoned to 2021. In 2021, the strawberries were harvested from the research trial and were tested for different quality parameters. The data were collected and were analyzed. Samples were sent to both collaborating institutions Delaware State (DSU) and Tennessee State Universities. The data were cleaned, analyzed and reported. Objective 3: Scientific papers, one book chapter, extension publications and educational materials were prepared, published. The results were dessiminated in field days and extension agent training sessions. Techncians and students, and postdoc were trained. The results were published and disseminated in extension agent training sessions and through presentations at symposiums and field days.

Publications

Type: Journal Articles Status: Published Year Published: 2021 Citation: Taghavi T, Patel H, Rafie R, 2021, Comparing pH differential and methanol-based methods for anthocyanin assessments of strawberries, Food Science & Nutrition, 2021, 00:1-9, DOI: 10.1002/fsn3.2065.
Type: Journal Articles Status: Published Year Published: 2021 Citation: Taghavi T, Bahamdan, G, Bell M., Patel H, Taylor, E, Rafie R, 2021, Comparison of five essential oils on postharvest strawberry fruit quality, Acta Horticulture, 1309: 941-945. , DOI 10.17660/ActaHortic.2021.1309.134.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Taghavi T, 2021, Comparison of five essential oils on postharvest strawberry fruit quality, 9th International Strawberry Symposium, ISHS, Oral presentation, Virtual, Italy 1-5 May 2021.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Taghavi T, 2021, VSU Field Day, Low Tillage Strawberry Raised Bed System, Presentations
Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Taghavi T, 2021, VSU Field Day, Application of clover cover crop to improve strawberry yield and quality, Presentations
Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2021 Citation: Taghavi T, 2021, VSU Field Day, High Tunnel Soilless Strawberry System Presentations
Type: Journal Articles Status: Published Year Published: 2020 Citation: Taghavi T, Bell M, James C, Taylor E, and Opoku M, Rafie R, 2020, Response of strawberries exposed to Thymol and Carvacrol vapors, Indian Journal of Horticulture, 77(3): 541-547.
Type: Book Chapters Status: Published Year Published: 2019 Citation: Taghavi, T, R. Siddiqui, L Rutto, The effect of preharvest factors on strawberry fruit quality, In: Strawberry, IntechOpen publications, 2019.
Type: Journal Articles Status: Published Year Published: 2022 Citation: Taghavi, T., Patel, H., & Rafie, R. (2022). Anthocyanin Extraction Method and Sample Preparation Affect Anthocyanin Yield of Strawberries. Natural Product Communications, 17(5), 1934578X221099970.
Type: Journal Articles Status: Published Year Published: 2022 Citation: Taghavi, T., Patel, H., Akande, O. E., & Galam, D. C. A. (2022). Total Anthocyanin Content of Strawberry and the Profile Changes by Extraction Methods and Sample Processing. Foods, 11(8), 1072.
Type: Journal Articles Status: Published Year Published: 2023 Citation: Patel, H., Taghavi, T., & Samtani, J. B. (2023). Fruit Quality of Several Strawberry Cultivars during the Harvest Season under High Tunnel and Open Field Environments. Horticulturae, 9(10), 1084.
Type: Journal Articles Status: Published Year Published: 2023 Citation: Taghavi, T., Patel, H., & Rafie, R. (2023). Extraction Solvents Affect Anthocyanin Yield, Color, and Profile of Strawberries. Plants, 12(9), 1833.
Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Taghavi T, H. Patel, 2023, Extracting Strawberry anthocyanins, testing different variables. Acta Hort., 1340: Accepted and Under Publication
Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Taghavi, T., C. James, M. Bell, E. Taylor, and C. Kim, 2019, Strawberry fruit decay is affected by plant volatiles, ISHS Vth International symposium on Postharvest Pathology, li�ge, Belgium, March 18-25, 2019, oral
Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: 1- James C., T. Taghavi, E. Taylor, M. Bell and C. Kim, 2019, Plant Volatiles: Can They Extend the Shelf Life and Reduce Fruit Decay of Strawberries (Fragaria � ananassa)? Association of Research Directors (ARD) Research Symposium 2019, Jacksonville, FL, March 30-April 3, oral.
Type: Journal Articles Status: Published Year Published: 2020 Citation: 3- Taghavi T, Bell M, Patel H, Opoku M and Rafie R, 2020, Response of Strawberries to Postharvest Application of Plant Volatiles, oral presentation, ASHS annual meeting, Virtual meeting, Aug 10-13, 2020.
Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Novel Application and Cultivation Technology for Medicinal Plants, 2020, Coordinator: Yan Chen, Louisiana State University AgCenter, Moderator: Toktam Taghavi, Virginia State University, ASHS annual meeting, Orlando, FL Aug 10-13, 2020.
Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Taghavi, T., J. Samtani, D. Liu, 2023, Agronomic and Post-harvest performance of strawberry cultivars in the high tunnel and open-field production in southeast Virginia, Int. J. Fruit Sci., Submitted
Type: Other Status: Published Year Published: 2023 Citation: Taghavi, T, Berry Crops: Production, Cultivation, Breeding and Health Benefits, Horticulturae, Guest Editor of the Special Issue
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 1- Galam D, Akande O, Githinji L, Taghavi T, 2022, Cover crop performance for straebrry annual hill system, Oral, Association of Research Directors, 20th biennial research symposium, Symposium, 2-5 Apr 2022.
Type: Other Status: Published Year Published: 2021 Citation: 10- Taghavi T, 2021, The hatch fund report for Virginia, NorthEast 1836 (NE1836)
Type: Other Status: Published Year Published: 2021 Citation: 1- Taghavi T, 2021, Application of cover crops in strawberry production. Impact Statement, VSU
Type: Other Status: Published Year Published: 2022 Citation: 1- Taghavi T, Sun Y, 2022, Cultivation and Application of Medicinal Plants in Plant, Human and Animal Health and How They Are Affected By Climate Change, Organized workshop, Jul 31-Aug3 2022, ASHS.
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 1- Taghavi T,2022, Application of Essential Oils on Extending the Shelf Life of Strawberries, Oral in workshop, Jul 31-Aug3 2022, ASHS.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: 14- Dale, A., & Taghavi, T. (2021, May). Juvenile runnering in F1 hybrid strawberry and its effect on inflorescence numbers. In IX International Strawberry Symposium 1309 (pp. 7-12).
Type: Other Status: Published Year Published: 2022 Citation: 15- Taghavi, T., VSU Report for NIMSS HATCH Fund S294 Annual Meeting in 2022
Type: Other Status: Published Year Published: 2023 Citation: 1- Taghavi, T., VSU Report for NIMSS HATCH Fund S294 Annual Meeting in 2023
Type: Other Status: Published Year Published: 2022 Citation: 17- Taghavi, T., VSU Final Report for NIMSS HATCH Fund NE1836 Annual Meeting in 2022
Type: Other Status: Published Year Published: 2023 Citation: 1- Taghavi, T., VSU Report for NIMSS HATCH Fund NE1836 Annual Meeting in 2023
Type: Other Status: Published Year Published: 2023 Citation: 19- Taghavi, T., VSU Impact Statement, essential Oils affect expression of genes regulating fruit firmness in strawberries, 2023
Type: Other Status: Published Year Published: 2023 Citation: 20- Taghavi, T., ARD Research Impact Statements, Early research shows essential oils may improve fruit shelf life, 2023
Type: Conference Papers and Presentations Status: Under Review Year Published: 2024 Citation: 1- Taghavi T, Patel H., Strawberry anthocyanins, Extractions, Assessments, and Storage, Abstract for American Society for Horticultural Sciences 2023.
Type: Other Status: Published Year Published: 2023 Citation: 23- Samtani J, Taghavi T. Final Report for SCBGP-2019 Grant, Agronomic, Post-harvest, and Economic evaluation of strawberry cultivars in high tunnel and open field production, 2023.
Type: Conference Papers and Presentations Status: Under Review Year Published: 2024 Citation: 24- Taghavi T, Patel H., Extracting strawberry anthocyanins, testing different variables, Abstract for Asian Horticultural Congress, ISHS 2023.

Progress 12/15/21 to 12/14/22

Outputs
Target Audience:Training in the lab and research filed: 1-Two technicians and one Postdoc were trained to do research literature, develop the experimental design (both at the field and in the lab), conduct lab analysis, collect, summarize and analyze data and prepare drafts. 2-The postdoc was sent for extra training to Delaware State University for three weeks and learned techniques on transcriptome analysis (as suggested in the proposal). 3-Four undergraduate students were trained in the field and the lab to do research literature review, develop the experimental design, conduct lab analysis, collect, summarize and analyze data and prepare paper drafts. Two of them presented posters in ARD symposium (Association for Research Directors of HBCUs, 2022help conducting the project. Formal classroom instructions, Laboratory instruction training One formal class for graduate students (Plant Morphology-BIOL 521) was taught for the Fall semester 2021-2022 and six master students were trained on the new and cutting edge knowledge in Plant Morphogenesis. Extension through presentations One presentation were made at VSU Field day for about 200 attendees including growers and other stakeholders (titles: High tunnel soilless strawberry system'). One oral presentation were made virtually at the 9th International Strawberry symposium in Italy. Many attended the meeting from all over the world. A workshop was organized on 'Application and production of medicinal plants for human, plant and animal health' at American Society for Horticultural Science (ASHS) annual general meeting. About 20 people attended in-person and many virtually. Changes/Problems:There is no major change to the project except that due to the pandemic and later start date, we were not able to start the experiment on time and it has been delayed for two years. Below are the challenges we are facing The postdoc has left the project early, due to the termination of his visa and not being eligible to work any more. The hiring process of technicians is very lengthy at Virginia State University and due to the current situation with the job market, we were not be able to hire a technician for the this year. However, we will continue our hiring efforts to find an hourly technician and finish all the activities. This year we were not able to acquire two of the promised strawberry varieties from nurseries; therefore, the project included only three out of five varieties. Due to the Covid 19, students had to quarantine when they were sick and this delayed the project. All activities were slower than the normal situation due to the pandemic lingering effects. What opportunities for training and professional development has the project provided?Two technicians and one postdoc were trained. The postdoc was also sent to DSU for supplemental training as suggested in the proposal. We have developed several laboratory protocols to assess the fruit and nutritional quality in our lab, such as anthocyanin assessments, fruit color, acidity, total soluble content, titratable acidity, total antioxidant and total polyphenols in strawberries. Two technicians and a postdoc were involved in almost all parts of these efforts and received training. Four undergraduate students were also trained and disseminated the research results in the scientific symposiums. How have the results been disseminated to communities of interest?The results were disseminated through formal classroom, Laboratory instructions, and presentations at the local and national symposiums. Results were also disseminated to the 200 growers, extension agents and many other stakeholders who attended the VSU field day in August 2022. What do you plan to do during the next reporting period to accomplish the goals?We are planning to finish activities in all three objectives. We will continue to finalize the data collection, and monitor the changes in fruit quality and senescence after harvest and during storage through transcriptome analysis. We train more students and technicians and continue to use innovative technologies such as gene expression analysis and proteomics studies to accomplish the goals. In developing the protocols, we plan to develop methods to assess total radical scavenging capacity.

Impacts
What was accomplished under these goals? Objective 1: Fruit color is an important factor in assessing fruit quality. Six methods have been developed and compared to assess anthocyanins in strawberry fruits. The extracted pigments were sent to a lab for HPLC analysis and identification of the pigment. Three manuscripts were published from this research and one is under development. Objective 2: The strawberries were harvested from the research trial and were tested for different quality parameters. The data were collected and will be analyzed in the coming months. Most of the samples sent to the collaborating institutions Delaware State (DSU) and Tennessee State Universities were analyzed and the data will be cleaned, analyzed and reported. Objective 3: Scientific papers, extension publications and educational materials were prepared and published. The results were disseminated in extension agent training sessions and through presentations at symposiums and field days. Two technicians, one postdoc and four undergraduate students were trained.

Publications

Type: Journal Articles Status: Published Year Published: 2022 Citation: 1- Taghavi, T., Patel, H., & Rafie, R. (2022). Anthocyanin Extraction Method and Sample Preparation Affect Anthocyanin Yield of Strawberries. Natural Product Communications, 17(5), 1934578X221099970
Type: Journal Articles Status: Published Year Published: 2022 Citation: 2- Taghavi, T., Patel, H., Akande, O. E., & Galam, D. C. A. (2022). Total Anthocyanin Content of Strawberry and the Profile Changes by Extraction Methods and Sample Processing. Foods, 11(8), 1072.
Type: Journal Articles Status: Published Year Published: 2021 Citation: 3- Taghavi T, Bahamdan, G, Bell M., Patel H, Taylor, E, Rafie R, 2021, Comparison of five essential oils on postharvest strawberry fruit quality, Acta Horticulture, 1309: 941-945. , DOI 10.17660/ActaHortic.2021.1309.134.
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 1- Galam D, Akande O, Githinji L, Taghavi T, 2022, Cover crop performance for straebrry annual hill system, Oral, Association of Research Directors, 20th biennial research symposium, Symposium, 2-5 Apr 2022.
Type: Other Status: Published Year Published: 2021 Citation: 2- Taghavi T, 2021, The hatch fund report for Virginia, NorthEast 1836 (NE1836)
Type: Other Status: Published Year Published: 2021 Citation: 3- Taghavi T, 2021, Application of cover crops in strawberry production. Impact Statement, VSU
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 4- Taghavi T, Sun Y, 2022, Cultivation and Application of Medicinal Plants in Plant, Human and Animal Health and How They Are Affected By Climate Change, Organized workshop, Jul 31-Aug3 2022, ASHS
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: 5- Taghavi T,2022, Application of Essential Oils on Extending the Shelf Life of Strawberries, Oral in workshop, Jul 31-Aug3 2022, ASHS.
Type: Journal Articles Status: Published Year Published: 2021 Citation: 6- Dale, A., & Taghavi, T. (2021, May). Juvenile runnering in F1 hybrid strawberry and its effect on inflorescence numbers. In IX International Strawberry Symposium 1309 (pp. 7-12).

Progress 12/15/20 to 12/14/21

Outputs
Target Audience:Due to the Covid 19 pandemic students were not allowed into the buildings and research farms, therefore, no students were trained this reporting year in the lab and research field. Two technicians and one Postdoc were trained to do research literature, develop the experimental design (both at the field and in the lab), conduct lab analysis, collect, summarize and analyze data and prepare drafts. The postdoc was sent for extra training to Delaware State University for three weeks and learned techniques on transcriptome analysis (as suggested in the proposal). One formal class for graduate students (Plant Morphology-BIOL 521) was taught for the Fall semester 2021-2022 and six master students were trained on the new and cutting edge knowledge in Plant Morphogenesis. Three presentations were made at VSU Field day for about 100 attendees including growers and other stakeholders (titles: 'Low tillage strawberry raised bed system', 'Application of clover cover crop to improve strawberry yield and quality', High tunnel soilless strawberry system'). One presentation was made at the HBCU/MSI Research Summit at Virginia Tech University on increasing collaboration among universities about 50 people attended the event. One oral presentation was made virtually at the 9th International Strawberry symposium in Italy. Many attended the meeting from all over the world. A workshop was organized on 'Immune-Improving Medicinal Plan Supplements and Challenges in the time of a Pandemic' at American Society for Horticultural Science (ASHS) annual general meeting. About 20 people attended in-person and many virtually. Changes/Problems:There is no major change to the project except that due to the pandemic and later start date, we were not able to start the experiment on time and it has been delayed for two years. Below are the challenges we are facing Two technicians have found other positions outside of VSU and separated from this project and VSU. The hiring process of technicians is very lengthy at Virginia State University and due to the current situation with the job market, we may not be able to hire a technician on time. However, we will advertise and start the hiring process again. This year we were not able to acquire one of the promised strawberry varieties from nurseries; therefore, the project included only four out of five varieties. Due to the Covid 19, students were not allowed to work in the lab or the research field. All activities were slower than the normal situation. What opportunities for training and professional development has the project provided?Two technicians and one postdoc were trained. The postdoc was also sent to DSU for supplemental training as suggested in the proposal. We have developed several laboratory protocols to assess the fruit and nutritional quality in our lab. We have developed protocols to assess anthocyanins, total antioxidant and total polyphenols in strawberries. Technicians and a postdoc were involved in almost all parts of these efforts and received training. How have the results been disseminated to communities of interest?The results were disseminated through formal classroom, laboratory instructions, and presentations at the local and national symposiums. Results were also disseminated to 30 growers, extension agents and many other stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We are planning to finish activities in all three objectives. We will continue to develop more laboratory protocols to assess the changes in fruit quality and senescence after harvest and during storage. We train more students and technicians and continue to use innovative technologies such as hyperspectral image, gene expression analysis and proteomics studies to accomplish the goals. In developing the protocols, we plan to develop methods to assess total radical scavenging capacity and early detection of diseases before they are visible to the naked eye.

Impacts
What was accomplished under these goals? Objective 1: Fruit color is an important factor in assessing fruit quality. Six methods have been developed and compared to assess anthocyanins in strawberry fruits. The extracted pigments were sent to a lab for HPLC analysis and identification of the pigment. One manuscript is under development and one was submitted and is under review. Objective 2: The strawberries were harvested from the research trial and were tested for different quality parameters. The data were collected and will be analyzed in the coming months. Samples were sent to both collaborating institutions Delaware State (DSU) and Tennessee State Universities. Objective 3: Scientific papers, extension publications and educational materials were prepared and published. The results were disseminated in extension agent training sessions and through presentations at symposiums. Technicians and one postdoc were trained. Mentoring: Two technicians and a postdoc were mentored through the course of this project. Equipment: No equipment was purchased. Collaborations: Through the course of the grant, several collaborations were developed. The first was with Dr. Jayesh Samtani to study the effect of different varieties on strawberry fruit quality parameters, including the effect of organic horticulture on anthocyanin in strawberries. The second was with Dr. Behnam Khatabi to test the isolated diseases effective on postharvest diseases of commodities. A third collaborative effort was undertaken with Dr. Nasser Ghariban from the Engineering Department at VSU and Dr. Huda Al-Ghaib from the industry (Commonwealth Center for Advanced Manufacturing; CCAM) to continue training an intern and develop methods to use hyperspectral images for early identification of strawberry diseases. A fourth collaboration was developed with Dr. Kalapalatha Melmaiee from Delaware State University to study the strawberry breeding germplasms for fruit quality during storage.

Publications

Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Taghavi T, Patel H, Rafie R, 2021, Comparing pH differential and methanol-based methods for anthocyanin assessments of strawberries, Food Science & Nutrition, 2021, 00:1-9, DOI: 10.1002/fsn3.2065.
Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Taghavi T, Bahamdan, G, Bell M., Patel H, Taylor, E, Rafie R, 2021, Comparison of five essential oils on postharvest strawberry fruit quality, Acta Horticulture, 1309: 941-945. , DOI 10.17660/ActaHortic.2021.1309.134.
Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Anthocyanin assessment methods (Submitted, under review), Fruits Journal
Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2021 Citation: Taghavi T, 2021, Comparison of five essential oils on postharvest strawberry fruit quality, 9th International Strawberry Symposium, ISHS, Oral presentation, Virtual, Italy 1-5 May 2021.
Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2021 Citation: Taghavi T, 2021, VSU Field Day, Low Tillage Strawberry Raised Bed System, Presentations
Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2021 Citation: Taghavi T, 2021, VSU Field Day, Application of clover cover crop to improve strawberry yield and quality, Presentations
Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2021 Citation: Taghavi T, 2021, VSU Field Day, High Tunnel Soilless Strawberry System Presentations

Progress 12/15/19 to 12/14/20

Outputs
Target Audience: Training in the lab New Virginia Cooperation Extension (VCE) agents visited the lab. On 10/17/2019. A short presentation was given to about 20 agents about the activities and research projects in the lab. Two graduate students were trained for two sessions on research project that was running in the lab. One intern was trained through collaboration with Engineering Department at VSU and industry (CCAM) to develop algorithms and machine learning techniques to use hyperspectral image data for early identification of strawberry diseases. Three technicians, one graduate and one undergraduate students and one research rotation student were trained to do research literature, develop the experimental design (both at the field and in the lab), conduct lab analysis, collect, summarize and analyze data and prepare drafts. Formal classroom instructions, Laboratory instruction training One formal class (11/6/2019) session was given to student of BIOL130 and one formal class (11/25/2019) session to students of AGRI140 class. Each class had about 20 students on average. Two laboratory (11/1/2019 and 11/6/2019) training sessions were given to eleven students of the course BIOL 310 (Plant Morphology) Two laboratory sessions (4/11 and 4/18/2019) training were given to ten student of the BIOL 418 (Plant physiology) class on using essential oils to improve postharvest fruit quality of strawberries Two sessions of formal classroom training were held (on 10/22/2018 and 9/17/2019) for students of AGRI 140 (Intro to Sustainable Ag & Society) and about 40 students participated in each session. Extension through presentations Presentation at American Society for Horticultural Science (ASHS) annual general meeting on "Response of Strawberries to Postharvest Application of Plant Volatiles" Changes/Problems:There is no major change to the project except that due to the pandemic and lockdown, we were not able to harvest any crop from our research plot. Below are the challenges we are facing The hiring process of the post doc is very lengthy at Virginia State University. The second postdoc was shortlisted and herapplication was sent to HR. We were waiting for the hiring process for 6 months. Then the postdoc did not accept the position because she found another position and we need to start the hiring process all over again. We have requested from the Director of our department a research plot to conduct the research. However, we have not been assigned a research plot yet. The first two years of the project we have borrowed a small piece of land from a colleague. This year we had to repeat the planting because the strawberries that we bought from an accredited nursery had anthracnose disease. We lost four cultivars out of five planted. Due to the Covid 19, the campus shut down on March 30th for three months. Strawberries were ready for harvest April 1st. Therefore, we lost the harvesting window of strawberries. We are not still allowed to work full time and are working in half capacity. Even so, the campus shut down twice for 14 days due to reports of department members that had Covid 19 or were in contact with someone with Covid 19. What opportunities for training and professional development has the project provided?Three technicians were trained and one undergraduate and one graduate students received training. We have developed several laboratory protocols to assess the fruit and nutritional quality at our lab. We have developed protocols to assess anthocyanins, total antioxidant and total polyphenols in strawberries. Technicians and students were involved in almost all parts of these efforts and received training. How have the results been disseminated to communities of interest?The results were disseminated through formal classroom, Laboratory instructions, and presentation at the local and national symposiums. Results were also disseminated to the 30 extension agents during in-service training. What do you plan to do during the next reporting period to accomplish the goals?We are planning to hire a postdoc and continue to develop more laboratory protocols to assess the changes in fruit quality and senescence after harvest and during storage. We train more students and technicians and continue to use innovative technologies such as hyperspectral image, gene expression analysis and proteomics studies to accomplish the goals. In developing the protocols, we plan to develop methods to assess total radical scavenging capacity and early detection of diseases before being visible by naked eyes.

Impacts
What was accomplished under these goals? Objective 1: Fruit color is an important factor in assessing fruit quality. Six methods have been developed and compared to assess anthocyanins in strawberry fruits. Two manuscript was submitted and is under review. Objective 2: Due to the pandemic and lockdown at the campus, no strawberry fruit could be harvested form the experimental plot. Therefore, these activities could not be performed and will be done next year. Objective 3: Scientific papers, extension publications and educational materials were prepared and published. The results were disseminated in extension agent training sessions and through presentations at symposiums. Technicians and students were trained and there is a plan to hire a postodoc.

Publications

Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Taghavi T, Bell M, James C, Taylor E, and Opoku M, Rafie R, 2020, Response of strawberries exposed to Thymol and Carvacrol vapors, Indian Journal of Horticulture, 77(3): 541-547.
Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Comparing PH differential and methanol (Submitted, under review), J of Food Science and Nutrition
Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Anthocyanin assessment methods (Submitted, under review), Fruits Journal
Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Taghavi T, Bell M, Patel H, Opoku M and Rafie R, 2020, Response of Strawberries to Postharvest Application of Plant Volatiles, oral presentation, ASHS annual meeting, Virtual meeting, Aug 10-13, 2020.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Novel Application and Cultivation Technology for Medicinal Plants, 2020, Coordinator: Yan Chen, Louisiana State University AgCenter, Moderator: Toktam Taghavi, Virginia State University, ASHS annual meeting, Orlando, FL Aug 10-13, 2020.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Comparison of five essential oils, ISHS, abstract accepted, IX International Strawberry Symposium.

Progress 12/15/18 to 12/14/19

Outputs
Target Audience:Training in the lab One intern was trained through collaboration with Engineering Department at VSU and industry (CCAM) to develop algorithms and machine learning techniques to use hyperspectral image data for early identification of strawberry diseases. Two technicians, three graduate and one undergraduate student and one research rotation student were trained to research literature, develop the experimental design (both at the field and in the lab), conduct lab analysis, collect, summarize and analyze data and prepare drafts. Formal classroom instructions, Laboratory instruction training A formal class (8/29/2018) and a laboratory (9/5/2018) training was given to eleven students of the course BIOL 310 (Plant Morphology) Two laboratory sessions (4/11 and 4/18/2019) training were given to ten students of the BIOL 418 (Plant physiology) class on using essential oils to improve postharvest fruit quality of strawberries Two sessions of formal classroom training were held (on 10/22/2018 and 9/17/2019) for students of AGRI 140 (Intro to Sustainable Ag & Society) and about 40 students participated in each session. Field tour On August 28th, 2019 the AgFest Field Day was held at the Randolph Research Farm and about 30 visitors were provided with information on the research projects and results. Extension through presentations Presentation at ISHS Vth International symposium on Postharvest Pathology, liège, Belgium, March 18-25, 2019, titled: "Strawberry fruit decay is affected by plant volatiles" Presentation at Association of Research Directors (ARD) Research Symposium 2019, Jacksonville, FL, March 30-April 3, 2019, titled: Plant Volatiles: Can They Extend the Shelf Life and Reduce Fruit Decay of Strawberries (Fragaria × ananassa)? Changes/Problems: The start date of the project is December, however, the first activity is planting strawberries in the field which should bedone in September. Therefore, in the first year of the experiment, we did not have strawberry varieties that we planned in the proposal and we used other strawberry varieties and developed the protocols. Therefore, we may need to apply for a no-cost extension, if necessary. The hiring process of the post doc is very lengthy at Virginia State University. A postdoc was shortlisted and his application was sent to HR. We were waiting for the hiring process for 4-5 months, before he found another position and we need to start the hiring process all over again. What opportunities for training and professional development has the project provided?Two technicians were trained and one undergraduate and three graduate students are receiving training. We have developed several laboratory protocols to assess the fruit and nutritional quality at our lab. We have developed protocols to measure sugar, titratable acidity, vitamin C, fungal contamination and ethylene. We also have developed methods to assess total anthocyanin, total antioxidant and total polyphenols in strawberries. Technicians and students were involved in almost all parts of these efforts and received training. How have the results been disseminated to communities of interest?The results were disseminated through formal classroom, Laboratory instructions, field day and presentation at the local and national symposiums. Results were also disseminated to 30 extension agents during in-service training. What do you plan to do during the next reporting period to accomplish the goals?We are planning to hire a postdoc and continue to develop more laboratory protocols to assess the changes in fruit quality and senescence after harvest and during storage. We willtrain more students and technicians and continue to use innovative technologies such as hyperspectral image, gene expression analysis and proteomics studies to accomplish the goals. In developing the protocols, we plan to develop methods to assess total radical scavenging capacity and early detection of diseases before being visible by naked eyes.

Impacts
What was accomplished under these goals? Objective 1:Five essential oils were screened for their efficacy against strawberry pathogens by placing 30 ul of them on a cotton ball inside the containers. Although most of them were effective in controlling the disease compared to control (no treatment), however two of them had less after-taste flavor. Also, efficacy of essential oils to reduce microbial load on fruit surface was tested. The first year results were notconclusive and more testing is requied to make any definitive determination. Objective 2 will be done in year 2 &3. Objective 3: Scientific papers, one book chapter, extension publications and educational materials were prepared and published. The results were dessiminated duringfield days and extension agent training sessions. Techncians and students were trained and there is a plan to hire a postodoc.

Publications

Type: Book Chapters Status: Published Year Published: 2019 Citation: Taghavi, T, R. Siddiqui, L Rutto, The effect of preharvest factors on strawberry fruit quality, In: Strawberry, IntechOpen publications.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Taghavi, T., C. James, M. Bell, E. Taylor, and C. Kim, 2019, Strawberry fruit decay is affected by plant volatiles, ISHS Vth International symposium on Postharvest Pathology, li�ge, Belgium, March 18-25, 2019, oral.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: James C., T. Taghavi, E. Taylor, M. Bell and C. Kim, 2019, Plant Volatiles: Can They Extend the Shelf Life and Reduce Fruit Decay of Strawberries (Fragaria � ananassa)? Association of Research Directors (ARD) Research Symposium 2019, Jacksonville, FL, March 30-April 3, oral.