LOUISIANA DEPARTMENT OF AGRICULTURE & FORESTRY MIKE STRAIN DVM, COMMISSIONER
Louisiana Specialty Crop Program Final Performance Report Agreement # 12-25-B-1464 January 8, 2016
CONTACTS
Program Administration: Michelle Estay, Director of Commodity Promotion & Research Louisiana Department of Agriculture & Forestry 47076 N. Morrison Blvd. Hammond, LA 70401-7308
Financial Officer: Dane Morgan, Assistant Commissioner Office of Management & Finance Louisiana Department of Agriculture & Forestry P.O. Box 3481 Baton Rouge, LA 70821-3481
CONTENTS
Abstract ...... 2
Project One...... 2
Project Two ...... 7
Project Three ...... 52
Project Four………………………………………………………………… 65
Project Five…………………………………………………………………. 76
Project Six………………………………………………………………….. 84
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PROGRAM OVERVIEW
The Louisiana Department of Agriculture and Forestry (LDAF) was awarded $351,115.72 in funding for the FY 2012 Specialty Crop Block Grant Program (SCBGP). LDAF implemented projects to enhance the competitiveness of specialty crops throughout the state.
Louisiana’s projects focused on programs working to inform consumers of the availability of Louisiana specialty crops, where they can be purchased for increased sales and consumption, specific specialty crop research to improve pest management and molecular analysis to assist in crop development and yield, and the study and promotion of Louisiana landscape plants.
These projects were chosen for their importance to Louisiana’s specialty crop industries and to help add money into the local economy. LDAF projects were designed to improve the competitiveness of Louisiana’s specialty crops and educate the consumer.
LDAF staff monitored each project by requiring quarterly activity reports and maintaining periodic phone calls, site visits and email update discussions. All invoicing and grant fund payments were completed.
PROJECT ONE: Consumer Awareness Campaign to Promote Buying Fresh Local Specialty Crop Produce
Louisiana Department of Agriculture and Forestry
Project Summary
This project’s focus was to market to the consuming public as to the availability of specialty crops in Louisiana and educate them on how easy it is to prepare and purchase Louisiana grown specialty crops. This project to promote buying fresh specialty crops, built on a prior year grant in which a rolling billboard was developed and constructed to promote and encourage consumers to eat fresh, local specialty crop produce. This promotion used a unique technique to reach consumers in their local areas by way of a traveling billboard that could be reused continuously at fairs and festivals, as well as, other venues throughout Louisiana.
This project built on the initial investment and activities by using the same rolling billboard and attending additional events reaching additional consumers in numerous parishes throughout Louisiana to promote and encourage consumers to eat fresh, local specialty crop as well as increase public awareness of the nutritional value of enjoying fresh, great tasting specialty crops and their availability.
The rising costs of radio, print, and commercial advertising has continued to increase over recent years. This makes it extremely hard to gain market saturation of messaging with limited dollars. The project provided LDAF the opportunity to reach consumers in mass numbers economically in a new memorable way.
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Project Approach
Using the previously construct rolling billboard LDAF staff met to determine the events that would be scheduled throughout Louisiana in years 1, 2 and 3. LDAF staff scheduled, registered for and participated in 21 parades to reach consumers with our specialty crop message.
During this process, it was determined that since the message was to eat healthy, fresh specialty crop produce, when it was possible to purchase fresh produce in season, we would pass out Louisiana specialty crops which included satsumas, oranges, cabbage, potatoes etc. During other times of the year collateral materials designed with our messaging and website information was distributed to drive the public to our website for more information and recipes. The resulting viewer website visits were used to measure the effects of the promotional campaign efforts.
LDAF staff worked to develop the promotional collateral materials with messaging placement. Materials were purchased. It was determined that much of the artwork and messaging that was created during the prior year grant project could be utilized to develop the needed items for this project’s activities; therefore, the dollars budgeted for an advertising agency was deemed unnecessary. The funds reserved for this expense was utilized to purchase additional materials to reach the consuming public in attendance at the events, which was a larger number than anticipated.
LDAF monitored website analytics reports prior to or post events to determine the activity driven to the site as a result of the project activities.
Goals and Outcomes Achieved
The objective of the program was to conduct a consumer awareness campaign to promote consumption of fresh local specialty crops throughout Louisiana utilizing the rolling billboard in outdoor marketing efforts. This was accomplished.
There were two main goals for this project. The first goal was to attend at least 15 fair/festival parades and/or events during the three years of this project. The target was met and exceeded with the attendance of and participation in 21 events. Parades/festivals attended were the Washington Parish Fair 2012, Kentwood Mardi Gras 2013, Strawberry Festival 2013, Kenilworth Independence Parade 2013, St. Tammany Parish Fair 2013, Washington Parish Fair 2013, Cracklin’ Festival 2013, Natchitoches Christmas Parade 2013, Livonia Carnival Parade 2014, Strawberry Festival 2014, Tangipahoa Parish Fair 2014, Louisiana Cattle Festival 2014, Louisiana Cotton Festival 2014, Washington Parish Fair 2014, Natchitoches Christmas Festival 2014, Livonia Carnival Parade 2015, Strawberry Festival 2015, Irish, Italian, Islenos Parade 2015, Ruston Peach Parade 2015, Louisiana Shrimp and Petroleum Festival, and Louisiana Sugarcane Festival 2015.
The second goal was to increase consumer awareness of the availability of and the benefits of purchasing and consuming fresh local specialty crops through messaging and driving the public to the Louisianagrown.com website to increase interest and knowledge of where to purchase from local producers and gain access to different recipes on how to prepare specialty crops. The goal was to measure success by increasing website visit movement after the events by 20%. Those visits would be compared to pre or post week visits to determine the increase or lack of increase. Using the website visit data, comparing the weeks pre/post parade hits to those right after the parade 3 event, it was determined that the 20% goal was reached. When averaging all event results, there was an average of 30% increase measured for the three year project. We deem this project a tremendous success. LDAF has plans to continue this consumer awareness promotional avenue into the future.
Fair/Festival Event Date Measurable Increase (i)/Decrease (d) Washington Parish Fair October 17, 2012 6% (i)
Kentwood Mardi Gras January 26, 2012 59% (i)
Strawberry Festival April 13, 2013 41% (i)
Kenilworth Independence Day July 3, 2013 90% (i)
St. Tammany Pair Fair September 27, 2013 17% (i)
Washington Parish Fair October 16, 2013 32% (i)
Cracklin’ Festival November 9, 2013 36% (i)
Natchitoches Christmas Parade December 7, 2013 3% (d)
Livonia Carnival Parade March 2, 2014 35% (i)
Strawberry Festival April 12, 2014 20% (i)
Tangipahoa Parish Fair October 3, 2014 27% (i)
Louisiana Cattle Festival October 11, 2014 11% (i)
Louisiana Cotton Festival October 12, 2014 11% (i)
Washington Parish Fair October 15, 2014 25% (i)
Natchitoches Christmas Parade December 6, 2014 47% (i)
Livonia Carnival Parade February 15, 2015 3% (i)
Strawberry Festival April 11, 2015 68% (i)
St. Bernard Irish, Italian, Islenos Parade April 12, 2015 68% (i)
Ruston Peach Parade June 27, 2015 15% (i)
Louisiana Shrimp and Petroleum Festival September 6, 2015 34% (i)
Louisiana Sugarcane Festival September 27, 2015 9% (d)
Project’s Average 30.1% (increase) Increase Measured
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Beneficiaries
This project benefited all specialty crop producers in Louisiana as consumer awareness was increased. Louisiana’s 3,223 vegetable farmers, 85 sweet potato farmers, 272 citrus growers and other numerous strawberry, watermelon, fig, blackberry, blueberry, and peach producers. Louisiana consumers exposed to the promotion efforts and educational information related to consuming fresh specialty crop produce benefited. Using attendance records, more than 300,000 were directly reached at events with countless other extended family members being impacted by the exposure the consumer awareness messaging acquired by their family members, by additional free media coverage and also through access to the LouisianaGrown.com website.
Lessons Learned
We learned that this project created an extreme amount of buzz. Extra free media coverage was realized due to this novel promotional technique. Social media coverage of the rolling billboard and our messaging was posted throughout several different venues. We learned to capitalize on the interest of this novel idea and garnered additional opportunities to gain exposure to the consuming public at no cost.
We learned that more and more event are charging entry fees for the events so budgeting for this should be considered. Also, some travel should be budgeted for events that are held at the outer distances of the state. Over nights could be needed when events are early in the morning at long distances away. Due to the success of this project LDAF plans to continue with this type of campaign into the future.
Additional Information Rolling Billboard
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Project Contact: Michelle Estay, Commodity Promotion and Research Director Louisiana Department of Agriculture and Forestry 47076 N. Morrison Blvd. Hammond, La 70401 985-345-9483
PROJECT TWO – Molecular Analysis of Pecan Nut Development
SUBGRANTEE: USDA - ARS
Project Summary:
Pecan nut development is coordinated by the properly timed expression of specific genes during the process. Global climate changes may make future pecan cultivation more difficult. Eating pecan nuts has several beneficial health properties, but they are also included the list of foods that commonly cause food allergy, and food allergy incidence has been increasing over the past decade. For these and other reasons, we have for the first time characterized the temporal gene expression occurring during pecan kernel development to better understand the process at the molecular level. This kind of information is essential to agricultural improvements that have been attained for other important crops such as soy, corn, and cotton. Our project is especially timely given the rise in food allergy incidence as a public health concern, and the environmental changes that may threaten the future of pecan farming.
Project Approach:
This study was conceived and supervised by Dr. Chris Mattison and he was involved in all aspects of the project. Samples from developing Sumner cultivar pecan nuts were collected by
Dr. Charlie Graham (LSU) at various times during the growing season to isolate and characterize
the gene expression occurring within a developing pecan kernel. The ribonucleic acid (RNA) from
these samples was processed by Dr. Doug Hinchliffe (USDA-ARS) and sequenced using an
Illumina-based platform at the Virginia Bioinformatics Institute (VBI). We have cataloged and
7 characterized the gene expression with modern bioinformatics analysis performed by Dr. Robert
Settlage (VBI). Selected fatty acid metabolism and pecan allergen target genes were used for
RT-qPCR comparison studies in the Sumner and Desirable cultivars by Dr. Hinchliffe.
Our study has greatly increased our knowledge of pecan gene sequences, and we have identified roughly 133,000 predicted unique pecan gene transcripts (unigenes). We have characterized the expression timing of these genes and identified patterns of expression that allow us to correlate the expression of specific groups of genes. For example, our bioinformatics analysis has
established correlations between the expression of some pecan fatty acid metabolism and allergen genes. The peak expression of these genes occurs in a similar pattern and suggests that their functions may be coupled or is dependent upon similar transcriptional cues. We have created a publically available website with details of our study and other pecan allergen information. We have monitored unique and repeat visits to this new informational page, and have accumulated roughly 10% of the page views of the parental LSU Pecan Research and Extension Welcome Page over the past 8 months. We have compared the expression of a few select genes using RT-qPCR, and found correlations between fatty acid metabolism and allergen gene expression. This work is being translated into journal manuscript form and will be submitted to a scientific journal for peer-review and publication in the coming months. A draft of the manuscript is included with this report as Appendix A. We plan to submit the new pecan gene sequence collection to the National
Center for Biotechnology Information when the paper is accepted.
Goals and Outcomes Achieved:
The goal of this study was to provide the pecan industry with information on new genetic and
molecular markers important for pecan nut development. We have identified over 130,000
potential pecan genes that may be used in the future as molecular markers or genetic targets for
horticultural study and genetic improvement. We have identified genes involved in pecan
8 nutritional aspects such as those involved in fatty acid metabolism and antioxidant production.
Further, our study has provided a foundation of pecan gene sequence and expression that can be applied to future studies better understand the process of pecan kernel development and the response of pecan gene expression to environmental changes and agricultural practices. We have compared the gene expression of selected allergen and fatty acid metabolism genes from the
Sumner and Desirable cultivars using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In some instances there is good overlap between the 2 cultivars, but we would like to compare our findings using more cultivars.
We have presented the findings of our project at several important state and regional pecan grower and pecan-industry related meetings. Dr. Mattison presented an outline of the project and updated findings at the Tri-State Pecan Growers Conference (6-20-2013, Vicksburg, MS), the Western
Pecan Growers Association (WPGA) meeting (3-2-2015, Las Cruces, NM), and the National
Pecan Sheller’s Association (NPSA) meeting (3-11-2015, Savannah GA). Dr. Graham presented an outline of the project and updated findings at the Tri-State Pecan Growers
Conference (6-19-2015, Natchez, MS).
With the assistance of the LSU AgCenter Information Technology group we have created a new
pecan allergen information website
(http://www.lsuagcenter.com/en/our_offices/research_stations/Pecan/Features/Pecan+Allergy+In
fo/Pecan-Allergen-Research-.htm) at the LSU-Pecan Station website containing important pecan
allergen information regarding food allergy, pecan allergens, as well as a short synopsis of our
study and a link to the annotated transcriptome results.
Table 1. LSU AgCenter Website Usage Summary Pecan Allergen Pecan Pecan Pecan Allergen Page Hits Month Welcome Welcome Page Page Hits (Unique) Page Hits Hits (Unique)
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Jun‐14 354 226 NA NA Jul‐14 310 203 NA NA Aug‐14 264 175 NA NA Sep‐14 249 168 NA NA Oct‐14 347 217 NA NA Nov‐14 566 351 NA NA Dec‐14 503 330 NA NA Jan‐15 356 223 NA NA Feb‐15 358 219 6 2 Mar‐15 358 226 55 25 Apr‐15 365 237 75 58 May‐15 325 214 7 5 Jun‐15 244 165 34 23 Jul‐15 274 164 16 15 Aug‐15 207 136 8 7 Sep‐15 247 176 8 7 total Feb‐ 2378 1537 209 142 September % of page hits 100 100 8.8 9.2
We have monitored usage of this website for 8 months (February-September 2015) to increase
the number of entries in our data set. We were targeting a 10% increase in the number web site visits during the 7 months following the presentation of our findings as a measure for interest in pecan gene sequence related information. However, we did not have an existing webpage with pecan nut development and nut allergy content, so we are comparing the page views of our new page to those of the parental LSU Pecan Research and Extension Station Welcome Page
(http://www.lsuagcenter.com/en/our_offices/research_stations/Pecan/) over the past 8 months.
Using this metric, during February-September the pecan allergen information page nearly attained the targeted 10% of the page views of the parental pecan research station page views in
terms of both unique and repeat web page views (Table 1). There were unique 209 repeat pecan allergen page views, or 8.8% compared to the repeat views at the pecan welcome page, and 142
unique page views, or 9.2% compared to the unique pecan welcome page views.
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To further promote our findings, Dr. Graham has published an article in the June 2015 issue of
“Pecan South” trade journal (Vol. 48, No. 4) describing our pecan kernel transcriptome project
and providing a link to the LSU Pecan Allergen website we have created. The website continues
to be accessible. We plan to submit the manuscript that describes the temporal pecan kernel
transcriptome and highlights the expression of pecan genes involved in fatty acid metabolism
and those that are known to act as allergens for publication in 2016. Some additional
experiments are being performed to characterize the proximate analysis of the developing pecan
nuts we used for our transcriptome analysis, and when this data is collected we will include it in
the final manuscript for journal submission.
Beneficiaries:
The primary beneficiaries for this work include pecan growers, breeders, and other pecan scientists. The full benefit of this work will require continued work in the area of pecan horticulture, and will likely take many years to mature. The sequencing of 130,000 potential pecan genes and the increased exposure of this work at the LSU website are the primary quantitative outputs. This importance of this work can also be highlighted by the observation that it was noted by other pecan scientists was in a recent personal transmission following the
Texas Pecan Growers Association meeting in July. The pecan research field as a whole will benefit from continued sequencing and bioinformatics analysis experiments similar to this one.
Lessons Learned:
The primary research problem we had was with the purification and preparation of RNA from
the pecan kernel samples. We were able to attain the needed samples using a specialized protocol, but only with extra rounds of sample purification and preparation. One need is to develop better methods for nucleic acid purification from pecan nuts to solve this problem for
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future sequencing projects. Presentation of the sequence data and its applications was well met
at pecan stakeholder meetings, but future research in this area may need to be more focused on a
target problem for a specific group of stakeholders. While the scientific pecan research
community was aided by the study findings, I believe other stakeholders, such as growers,
understand the importance of this type of work, but want it to be translated into ‘real-life’
applications more quickly. Unfortunately, this type of foundation laying work takes time to be
applied to their farming practices. It is important to rapidly and efficiently follow-up this type of
work with future studies that apply the findings to specifically targeted horticultural and
environmentally applied studies.
Contact person: Christopher P Mattison, PhD USDA-ARS-SRRC 1100 Robert E Lee Blvd New Orleans, LA 70124 Office: 504-286-4392 Cell: 720-771-0306 Fax: 504-286-4419 E-mail: [email protected]
Additional information:
Appendix A contains the manuscript draft describing the experimental work. Attached file of Supplimental Table 1 and Table 2
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Appendix A
Characterization of allergen and lipid metabolism genes within a developing pecan (Carya
illinoinensis) embryo transcriptome
Christopher P Mattison1*, Ruhi Rai2, Robert E Settlage2, Doug J Hinchliffe1, John M Bland1,
Suzanne Brashear1, Crista Madison1, Charlie J Graham3, Matthew R Tarver4, Christopher
1 1 Florane , and Peter J Bechtel
1USDA-ARS, Southern Regional Research Center, 1100 Robert E Lee Blvd, New Orleans, LA,
70124, USA
2Virginia Bioinformatics Institute, 1015 Life Science Circle, Blacksburg, VA, 24061, USA
3Louisiana State University-AgCenter, Agricultural Experiment Station, Pecan Research &
Extension Station, 10300 Harts Island Road, Shreveport, LA 71115, USA
4Bayer CropScience, Biologics, 890 Embarcadero Drive, West Sacramento, CA 95605, USA
*Corresponding author:
Mail: Southern Regional Research Center, Agricultural Research Service, U.S. Department of
Agriculture, 1100 Robert E. Lee Blvd., New Orleans, LA 70124, USA. Phone: (504) 286-4392.
Fax: (504) 286-4419. E-mail: [email protected].
Running title: temporal pecan embryo transcriptome
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Abstract
The pecan nut is a nutrient rich part of a healthy diet full of good fatty acids and antioxidants, but can also cause allergic reactions in people suffering from food allergy to the nuts. We characterized the transcriptome of a developing pecan nut to identify the gene expression occurring during the process and to highlight those genes involved in fatty acid metabolism and those that commonly act as food allergens. Pecan samples were collected at several time points during the embryo development process including the water, gel, dough, and mature nut stages.
Library preparation and sequencing was performed using Illumina based mRNA HiSeq with
RNA from 4 time points during the growing season during August and September 2012. Sequence
analysis with Trinotate software following the Trinity protocol identified 133,000 unigenes with
52,267 named transcripts and 45,882 annotated genes. A total of 27,312 genes were defined by
GO annotation. Gene expression clustering analysis identified 12 different gene expression
profiles, each containing a number of genes. Three pecan seed storage proteins that commonly act as allergens, Car i 1, 4, and a 7S vicilin homolog were significantly upregulated during the time course. Some of the genes involved in lipid metabolism that we identified were found to have expression patterns similar to the allergen genes based upon our clustering analysis and independent qRT-PCR analysis. The sequence and gene expression information we have generated is a great resource and will provide a foundation for continued improvement of pecan cultivars and agricultural practices.
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Introduction
Pecan (Carya illinoinensis) species are native to North America and are an important agricultural crop in the US. The US leads the world in pecan nut production accounting for approximately
80% of total production with an estimated value for the 2011 US crop at close to $700 million (1).
Pecan nuts are valued for their nutritional characteristics providing protein and unsaturated fats, as well as their enjoyable sensory qualities. Nuts contain high levels of antioxidants that are beneficial to health (2), and pecan’s have been shown to contain very high levels of antioxidants
(3, 4). Since 1980, Americans have consumed roughly half a pound of pecans per year (5).
Pecan nut and other tree nut consumption has been associated with several health benefits including improved serum lipid profile (2, 6-14).
Unfortunately, while increased pecan nut consumption provides health benefits to most of the US
population, a small but increasing percentage suffers from tree nut allergies. Tree nuts are included in the FDAs list of eight major food allergens and foods containing allergens such as pecan nuts must be declared in the product label. The frequency of childhood tree nut allergy
has steadily risen over the past decade (15), and tree nut allergies are rarely outgrown (16). Most food allergy induced fatalities arise from accidental exposures (17, 18). There are 3 seed storage proteins that commonly act as tree nut and peanut allergens, 2S albumins, 7S vicilins, and 11S legumins (19).
Allergy to pecan nuts is prevalent and pecan allergens cross-react with walnut and hazelnut allergens (20, 21). There are reports describing the conserved seed storage protein pecan allergens including the 2S albumin Car i 1 (22), the 11S legumin Car i 4 (23), and the 7S vicilin
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(24). Pecan nut allergens, like other nut allergens, are generally resistant to processing steps aimed at reducing or eliminating their ability to cause allergy (25, 26). Further, there is a documented example of aged or heated pecan nuts specifically correlated to IgE binding and allergic reaction to pecan nut (27, 28). This suggests important changes occur within developing pecan nuts late in maturation and possibly during storage. How peanuts and tree nuts regulate allergen gene expression and accumulation within developing nuts is not well understood. There has been some work on allergen expression and regulation in peanut (29-32) as well as a report of pecan allergen gene expression during pecan kernel development (33).
Pecan and other nuts are good sources of healthy fats, and pecans are composed of 62-89% lipid
(34-36). Pecan oil is high in oleic and linoleic unsaturated fats and is only about 5-10% saturated fat (34-36). However, there is evidence that dietary lipids such as those founds in pecans and other tree nuts may act as adjuvants for allergic sensitization to seed storage proteins found in
nuts (37). For example, the Brazil nut 2S albumin required the presence of Brazil nut lipids to
induce antibody response in mice (38, 39). Other 2S albumins from sunflower and mustard have also been documented to associate with lipids (40-42). When and how fatty acids and nut allergens accumulate during kernel development likely depends upon the coordinated expression of genes that respond to nutritional and environmental factors that may influence the process.
Fatty acids consist of long carbon chains terminating in carboxylic acid groups. They perform multiple functions in plants including serving as an important source of energy reserves, essential membrane components, signaling molecules, and can play roles in plant defense (43, 44). Fatty acid synthesis is well characterized involving many enzymes including β-ketoacyl-ACP synthase
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III enzyme (GO:0033818) (45, 46), Beta-ketoacyl-CoA synthase I (GO:0004315) and II
(GO:0033817), stearoyl-ACP desaturase (GO:0045300), and Acyl-ACP thioesterase enzymes
(GO:0016297). There are likely multiple isoforms for each of the enzymes involved in fatty acid
metabolism in a given organism, and the regulated expression of the genes encoding these
enzymes continues to be an area of intense investigation (47-50).
Underlying pecan nut development is a well-orchestrated set of genetic factors that integrate environmental cues, nutritional state, and plant stress. The gene expression and protein accumulation within a developing nut determines the yield, quality, and nutritional value of the crop. Growth and development of pecan nuts has been separated into distinct physiological phases, and there are several reports describing pecan nut development at the tissue and physiological level (51-53). Expanding upon these earlier descriptions of the process, pecan nut development was documented as proceeding through three main phases (54). Phase 1 includes the appearance of pistillate flowers, pollination, fertilization, and nut growth up to initiation of the endosperm. At the start of Phase 2, pecan nuts are approx. 40-50% of their final width and length (55), and during this phase, usually during late-June through early-August, the size of the nut is determined. There is rapid growth of the ovule and nut body. This phase is initiated by
the development of the cellular endosperm and ends with the initiation of shell hardening at the
apex of the nut. Several factors, including environmental conditions and pests, can affect the size
of the nuts (56-58).
Phase 3 of nut development, usually from August-October, is characterized by lignification of the shell, growth and maturation of the embryo and the cotyledons, and dehiscence of the shuck
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from the nut. The endosperm develops into a thick succulent pad of material (54) which contains carbohydrates, but no fat (59). The embryo grows basipetally along the outer wall of the ovule, absorbing the endosperm, while the cotyledons continue to thicken until the nut reaches maturity, marked by separation of the involucres from the nut (54, 55). The mature kernel consists of approx. 70% (w/w) oil, 8% (w/w) protein, 8% (w/w) water, and 2% (w/w) sugars (34, 60).
When mature, the hull splits down four creases along the length of the seed pod to enable nut release.
While there are several descriptions of embryonic pecan nut development at the morphological
and tissue levels in this stage, a large gap remains in our understanding of the process at the
molecular level. This area of research has been largely dormant for the past 40 years, but advances in molecular technology allow us to expand upon these findings and more fully characterize this process. Past reports have provided an initial glimpse of the proteins from developing pecan seeds (61), and described a partial cDNA encoding a seed storage protein (62).
Characterization of pecan allergens has also provided some gene sequence information including that of the 11S legumin (Car i 4) and 2S albumin (Car i 1) allergens (22, 23). Previously we
have investigated pecan allergen gene expression in developing pecans and found that allergen mRNA levels peak in September during the dough stage, and then decline gradually as nut maturation progresses (33). Transcriptome studies can yield powerful information for a given biological process and can point to genes of interest and coordination of gene expression. We are most interested in the expression of allergen and fatty acid metabolism genes during the nut filling process. Specific to this report, we target the internal cavity (central vacuole) of the developing pecan kernel prior to and during the period of kernel development, and focus on the
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latter stage of nut development, characterizing the temporal gene expression occurring during
phase 3 of pecan kernel development. We characterize the timing of allergen and fatty acid metabolism genes to try to draw correlations between allergen and fatty acid accumulation in developing pecan nuts.
Materials and Methods
Pecan nut samples
Nut samples (n = 10) were collected based on morphology from individual ‘Desirable’ and
‘Sumner’ trees at the Louisiana State University Shreveport, LA at various dates from August
through September of 2012. Each tree received standard agronomic practices (63). Nuts were
collected at “late water” stage (August 11), “gel stage” (August 23), “dough stage” (September
4), and “mature nut” (September 20). All samples were frozen immediately in liquid nitrogen
and stored at –80ºC for later chemical analysis. Tissue samples, either liquid, gel, or kernel depending upon the time point, were collected from the internal nut cavity for RNA isolation.
The nut cavity tissues in our collection included the contents of the central vacuole, endosperm,
embryo, placenta, seed coat, ovary packing tissue, developing fruit, and the cotyledon lobes as depicted by McKay (52). We did not include structural components of the nut such as the hull, shell, and middle septum in this analysis.
Proximate analysis of developing pecan nut tissue
Total RNA isolation
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Frozen pecan nut cavity tissues were ground to a fine powder using a mortar and pestle and liquid nitrogen. Total RNA was isolated from pecan nut cavity tissues using a protocol using a protocol specifically developed for pine needles and other recalcitrant plant materials (64) with the following modifications. The amount of ground nut cavity tissue was 100 mg for each sample which was transferred to 1.7 ml microcentrifuge tubes. The volume of extraction buffer added to the tissue was 600 µl followed by 2 extractions with 600 µl chloroform:isoamyl alcohol (24:1).
The aqueous phase was transferred to a new 1.7 ml microcentrifuge tube and 175 µl of 8
M LiCl was added and the RNA precipitated overnight at 4°C. The remainder of the protocol
was followed as described by Chang et al., 1993. The concentration and purity of each RNA sample was determined using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies
Inc., Wilmington, DE) and a Qubit 2.0 fluorometer (Life Technologies, Grand Island, NY). The
RNA quality for each sample was determined by RNA integrity number (RIN) using an Agilent
Bioanalyzer 2100 and the RNA 6000 Nano Kit Chip (Agilent Technologies Inc., Santa Clara,
CA) with 250 ng of total RNA per sample.
Stranded RNA-seq library construction and sequencing
Isolated RNA from the Sumner cultivar was used for library construction and sequencing. All
sequencing library preparations were performed using an Apollo 324 Robot (Wafergen, CA,
USA). Total RNA quality was validated on an Agilent BioAnalyzer 2100 (Agilent
Technologies, Santa Clara CA, USA). A PrepX PolyA mRNA Isolation Kit (Wafergen,
Fremont, CA, USA) was used to enrich 250 ng of total RNA for polyA RNA. The enriched
PolyA RNA was then converted into a library of template molecules using a PrepX RNA-Seq for
Illumina Library Kit (Wafergen, Fremont, CA, USA) for subsequent cluster generation and
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sequencing by Illumina HiSeq. Briefly, poly-A mRNA was fragmented into smaller pieces
(~140nt). Adapters (5’ and 3’) were ligated to the cleaved RNA fragments and converted to first
strand cDNA using reverse transcriptase, followed by second strand synthesis. The products
were then purified and enriched with 13 cycles of PCR to create the final cDNA library. The generated 280-300bp libraries (containing 160-180 bp inserts) were validated using an Agilent
2100 Bioanalyzer and quantified using a Quant-iT dsDNA HS Kit (Invitrogen) and qPCR.
Individually indexed cDNA libraries (6 to 12) were pooled and sequenced on Illumina HiSeq in order to get a minimum of 30 million reads. Libraries were clustered onto a flow cell using
Illumina’s TruSeq PE Cluster Kit v3-cBOT-HS, and sequenced with 101 Paired-End Sequencing on a HiSeq 2500 Ultra-High-Throughput Sequencing System using a TruSeq SBS Kit v3-HS
(200-cycles).
Assembly, annotation, and gene expression analysis
Following sequencing, data was trimmed for both adaptor and quality using a combination of ea-
utils and Btrim (65, 66). Trimmed-paired reads were then de novo assembled using Trinity
software (67) as strand specific reads. Annotation of transcripts (including identification of ORFs and attaching both description and function) was performed using the Trinotate package
(http://www.vcru.wisc.edu/simonlab/bioinformatics/programs/trinity/docs/annotation/Trinotate.h
tml) using default parameters as described in the Trinotate protocol. Reads were then aligned to
the assembled transcriptome using Bowtie2 (68). HTSeq and DESeq2 were used to count and determine significance of gene expression changes as described in the Trinity abundance estimation and identifying DE feature protocols (69, 70). Cluster profile plots were created in R using kmeans to define 12 unique clusters of gene profiles.
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Quantitative real-time PCR (qRT-PCR)
The experimental procedures and data analysis related to RT-qPCR were performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (71). Isolated RNA from both Sumner and Desirable cultivars was used for RT-qPCR experiments. The cDNA synthesis reactions were performed using the iScript™ cDNA
Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's protocol with 1 μg of total RNA per reaction used as template. Control cDNA synthesis reactions to check for genomic DNA contamination during RT-qPCR consisted of the same template and components as the experimental reactions without the reverse transcriptase enzyme. The RT- qPCR reactions were performed with iTaq™ SYBR® Green Supermix (Bio-Rad Laboratories) in a Bio-Rad CFX96 real time PCR detection system. Thermal cycler parameters for RT-qPCR were as follows: 95°C 3 minutes, 50 cycles of 95°C 15 seconds, 60°C 30 seconds. A dissociation curve was generated and used to validate that a single amplicon was present for each
RT-qPCR reaction. The calculations for amplification efficiencies of the target and reference genes, RNA inhibition assays, and the relative quantifications of the different target gene
transcript abundances were performed using the comparative Cq method as described in the ABI
Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative
PCR (Applied Biosystems, Foster City, CA) with the following modification: the average of two reference gene Cq values was determined by taking the geometric mean which was used to calculate the ΔCq values for the individual target genes (72). The endogenous reference genes used in the RT-qPCR reactions were the C. illinoinensis 18S ribosomal RNA gene and the trnl-
22
tmF chloroplast intergenic region as utilized in a previous study (33). Primer pairs used for the qRT-PCR analysis are listed in Table 2.
Results
Moisture, protein, and fatty acid profile during pecan kernel development
In order to better characterize the latter third stage of pecan nut development from Sumner and
Desirable cultivars, we collected nuts at various times during the 2012 season. The time points were correlated to the water, gel, dough, and mature nut developmental stages (Figure 1), and we analyzed the samples for proximate analysis. Water content within the developing kernel decreased during kernel development and was inversely correlated to protein and lipid content
(Figure 2).
RNA sequence assembly and gene annotation
We sequenced the genes expressed within the kernel to try to correlate the physiological and biochemical developments within the developing kernel to changes in gene expression using 4 time points. RNA from four time points, August 11, August 23, September 4, and September 20,
2012, were evaluated using the Sumner cultivar samples. Sequencing was performed using
Illumina based mRNA HiSeq and 200ng of total RNA isolated from each developing pecan nut sample. We sequenced and assembled a total of 86,526,858 bases (40.4% GC, a maximum contig length of 12,160 bases, average length of 607 bases, see Table 1). ORF annotation was performed using the SwissProt/Uniprot/Uniref90 databases using Trinotate. Following the
Trinity protocol, we observe 142K transcripts collapsed into ca. 133K unigenes. Further attempts at clustering transcripts into genes (TGI-CL, CD-HIT-EST) did not result in an
23
appreciable reduction in gene number presumably due to issues in assembly arising from either sample quality or population polymorphisms. There were 45,882 named Trinity genes with annotation and 52,267 named Trinity transcripts.
Annotated genes were given GO category assignments (biological process, cellular component, and molecular function). A total of 27,312 genes received GO annotations including 836 unique cellular component, 2,355 unique molecular function, and 4,309 unique biological process annotations. We identified a large number of gene products functioning in biological processes such as carbohydrate and lipid metabolism, defense, stress response, and signaling; those localizing to or within membranes; and those involved in molecular functions related to ATP binding and protein serine/threonine kinase activity. The top 25 GO groups having significant temporal gene expression differences throughout the time course are represented in Figure 3, and they are plotted with respect to the number of genes within the biological process (3A), cellular component (3B), and molecular function (3C) categories. There were a large number of genes involved in transcription, components of the nucleus or membranes, and ATP binding during embryo development that stood out from these plots.
The relative magnitude and directional change in gene expression within each category was also
compared between time points, transition 1 (8/11-8/23), transition 2 (8/23-9/4), and transition 3
(9/4-9/20) (Figure 4 A-C). Amino acid transport, cellulose biosynthesis, mucilage extrusion, and lipid transport were among the biological processes that were generally up-regulated at each transition, while those associated with protein folding were generally down-regulated at each transition. Genes involved in transmembrane transport were up-regulated during transition 1 and
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3. Conversely, translation associated genes were up-regulated during the transition 2. As groups, anchored membrane components, anchored components of the plasma membrane, golgi membrane, and plasma membrane constituents were mostly up-regulated at each of the transitions, while monolayer-surrounded lipid storage body components and nucleolus components were down regulated at transition1 and 3, but up-regulated at transition 2. Lysosome
and microtubule associated proteins were up-regulated during transition 1 and 3, but down-
regulated during transition 2. Molecular functions that were generally up-regulated at each transition included acid phosphatase activity, cellulose synthesis, and serine-type endopeptidase activity. While these observations provide a broad view of gene expression changes, we were interested in focusing our analysis on allergen and fatty acid metabolism genes.
Allergen gene expression analysis
We inspected the mRNA sequencing data for temporal gene expression changes in the 3 conserved pecan allergens. The absolute expression values of each of the 3 allergen genes, Car i
1 (comp34075_c0), Car i 4 (comp34066_c0), and the 7S vicilin homolog (comp34067_c0) demonstrate that expression of the 7S vicilin increases the most during development (Figure 5A).
A great deal of the increase in expression of the 7S vicilin occurred during the first and third transitions. When the expression changes were scaled, clear differences in expression profiles of the allergens were easier to discern. While expression of each of the allergen genes increased at the first transition, the expression of the Car i 1 2S albumin continued to increase during the second transition. During this time, expression of the 7S vicilin homolog (comp34067_c0) and
Car i 4 (comp34066_c0) leveled off or decreased slightly. Further, there was a sharp decrease in
25
expression of the Car i 1 2S albumin gene (comp34075_c0) at the third transition as expression
of the Car i 4 (comp34066_c0) and 7S vicilin (comp34067_c0) genes was elevated at the same
time (Figure 5B).
Gene expression profile clustering
The 3 pecan allergen genes had different expression profiles, although the overall pattern of Car i
4 (comp34066_c0) and the 7S vicilin (comp34067_c0) genes were more similar. To identify groups of genes with comparable expression profiles, and identify those with expression patterns similar to the allergen genes we evaluated the gene expression data using a cluster analysis of scaled gene expression values. The three allergen genes were categorized into cluster 1
(comp34075_c0), 2 (comp34067_c0), and 3 (comp34066_c0) by this analysis (Figure 6). These
3 clusters (1-3) are similar in that there was an overall increase in expression with time. There were 233, 594, and 422 genes in clusters 1, 2, and 3 respectively, and we were able to annotate
80, 245, and 169 genes in clusters 1-3 respectively. Included in these clusters were numerous genes involved in DNA binding and transcription, transmembrane transport functions, nutrient reservoir activity, and lipid metabolism (Supplementary material Table 1). For example, the expression of several fatty acid metabolism and storage genes were identified in cluster 3 including acyltransferase (comp28848_c0), desaturase (comp17048_c0 and comp34232_c1), beta-ketoacyl-CoA synthase I (comp34322_c0), and oleosins (comp34102_c0 and comp34087_c0). The expression pattern of a couple keto-ACP synthase genes (comp38847_c0 and comp126782_c0) matched that of the other genes included in cluster 2. Genes in cluster 1 whose expression was similar to Car i 1, the 2S albumin, peaked at transition 2 (Figure 6).
Genes within cluster 1 that were up-regulated during transition 2 were involved in ethylene-
26
activated signaling, response to stress, as well as fatty acid metabolism (Supplementary material
Table 1). The expression pattern of a long-chain fatty acid-CoA ligase enzyme (comp39519_c0)
matched it to cluster 1.
We differentiated 9 other distinct expression profile clusters from the gene expression data
(Figure 6 and Sup Mat Table 1). For several of the clusters including 4,5,7,8 and 9, gene expression showed an overall decline (Figure 6), however the other cluster profiles had either maintained or increased overall gene expression. There were 455, 200, 741, 248, and 72 genes in clusters 4,5,7,8 and 9 respectively. The types of gene functions in each of the clusters that were up-regulated varied. For example, genes within cluster 10 (containing 120 genes) included those involved in ATP biosynthesis, cellulose biosynthesis, seed coat development, and xylem development (Figure 6 and Supplementary material Table 1). Genes in cluster 6 (containing 323 genes) and cluster 12 (containing 391 genes) showed an increase in expression during the first transition (Figure 6 and Sup Mat Table 1), and included genes involved in pollen and cell wall- related synthesis or function. Genes within cluster 6 and 11 (containing 278 genes) included those involved in stress response or defense, protein synthesis, transport functions, and fatty acid metabolism.
Expression of Genes Involved in Fatty Acid Metabolism
Pecan oil is highly nutritious and studies have provided support for the health benefits of pecan
consumption (7). Pecan oil contains very low levels of saturated fat, and is high in
monounsaturated oleic and polyunsaturated linoleic acids (73). We inspected the gene expression data to identify those annotated genes thought to be involved in fatty acid
27
metabolism. We identified 299 genes (52 whose expression was significantly altered) involved in
various steps of fatty acid metabolism (Sup Mat Table 2), and these had a wide range of expression profiles, some of which fit into the clusters described above. Several of these fatty acid metabolism genes were distinctly upregulated during the time course of nut filling we
studied. For example, there were 10 acyl carrier protein (ACP) encoding genes, and among them all but 2 were up-regulated during the time course. Seven of the ACP encoding genes had increased expression during the first transition, and in particular comp34310_co was elevated
~600%, the most based upon scaled expression changes. There were 3 malonyl-CoA:ACP transacylase genes, and one of these, comp40800_co, was upregulated ~100%, and much of this occurred during the third transition. There were 36 ketoacyl-ACP synthase genes, and many of them were down-regulated during the time course. Exceptions to this included comp38847_c0, comp126782, and comp91447_co which were each highly up-regulated (9,859, 4,564, and,
969% respectively).
Two hydroxyacyl-ACP dehydratase genes were identified, and comp40728_co and comp39981_co were both upregulated during the first transition. Comp39981_co remained elevated during transition 1, 2, and 3, while comp40728_co expression was elevated sharply during transition 1 and then steadily declined during transition 2 and 3. Two enoyl-ACP reductase genes were identified, comp25799_co and comp40412_co, but comp40412_co had the most noticeable change and was upregulated 205% during the first transition while comp25799_co was up 149%. Seven of the eleven acyl-CoA synthetase genes were downregulated during the time course, but comp33533_co, comp17321_co, comp39519_c0, and comp36497_co were exceptions. Comp39519_co, comp33533_co, and comp17321_co were up sharply during the first transition (627, 408, and 303% respectively), while comp36497_co was
28
relatively flat during transition 1 and 2, and was upregulated 274% during the third transition.
Only one of the two acyl-ACP thioesterase genes we identified, comp33673_co, was upregulated
during the time course with a marked increase of 513% occurring during the first transition.
There were 103 acyltransferase genes identified in our analysis and 47 were upregulated over the duration of the developmental period we examined. Of these 47, there were 7 genes including comp64068_co (19,154%), comp16498_co (3,348%), comp60602_co (2,624%), comp20325_co
(2,493%), comp122400_co (2,290%), comp22057_c0 (1,920%), and comp84270_c0 (1,174%) that were markedly upregulated. Three of the 38 desaturase genes we sequenced were
upregulated over 500% during the time course. Comp34232_c1 was up 2,299%, comp34153_co
was up 969%, and comp17048_co was up 722%. Another desaturase, comp17172_c0, had a sharp increase in expression during the first transition (1,539%) similar to comp34232_c1 and
comp34153_c0, but its level dropped substantially during the second and third transitions. Among the eight acetyl-CoA carboxylase genes we identified, comp34460_c0 (from cluster 1), comp17318_c0, and comp43181_c0 were upregulated. Comp34460_c0, previously highlighted in
cluster 1, and comp43181_c0 had a similar expression spike at the first transition, while comp17318_c0 showed a more steady increase over the time course. There were a pair of 3- oxoacyl-ACP synthase III genes and one of them, comp21054_c0, was upregulated sharply during
the first transition like the acetyl-CoA carboxylase comp34460_c0. We also identified four 3- oxoacyl-[acyl-carrier-protein] synthase II genes and comp34322_c0 had the largest (856%) change during the time course. There were two of five 3-oxoacyl-ACP reductase genes, comp37759_c0 and comp25546_c0, whose expression was upregulated, 855 and 636% respectively, over the time course. Notably, comp37759_c0 was upregulated 1,321% during the third transition. A third 3-oxoacyl-ACP reductase gene, comp34514_c0, was upregulated over
29
400% during the first transition, but its level decreased substantially during transition 2 and 3.
None of the five 3-hydroxyacyl-ACP dehydratase genes was substantially changed cumulatively during the time course, but comp comp40728_c0 was upregulated 344% during the first transition
and declined to initial levels after that. Both of the enoyl-ACP reductase I genes we identified had approximately 400% increased expression during transition 1, but declined rapidly to initial levels during transition 2 and 3.
qRT-PCR of selected pecan allergen and fatty acid metabolism gene expression
We selected 12 of the fatty acid metabolism genes we identified from the transcriptome sequencing and evaluated their gene expression using qRT-PCR as a secondary evaluation of their expression during pecan kernel development. We performed this analysis with samples from both the Sumner cultivar used for transcriptome analysis and a second cultivar, Desirable, for comparison (Figure 7). Each of these genes was significantly up-regulated at some point during the time course we examined, and for some including a 3-oxoacyl-ACP synthase III
(comp21054_c0), an acyl-[ACP] desaturase (comp34153_c0), an acyl-ACP thioesterase
(comp33673_c0), and an acyl carrier protein (comp34310_c0) there was a good correlation to the expression pattern observed using the transcriptome analysis.
There were a few distinct expression patterns revealed by this analysis. In general terms, the expression pattern of a hydroxyl-ACP dehydratase (comp39981_c0), an acyl-activating enzyme
(comp40417_c0), and a malonyl-CoA:ACP transcyclase (comp40800_c0) were alike as they all peaked at the first sampling time point in August (Figure 7). The comp40417_c0 expression data from our transcriptome analysis agreed well with the RT-qPCR data and showed a greater than
30
90% decrease in the first transition. The expression pattern of a 3-oxoacyl-ACP synthase III
(comp21054_c0), an acyl-ACP thioesterase (comp33673_c0), an acyl-[ACP] desaturase
(comp34153_c0), and an acyl carrier protein (comp34310_c0) assessed by RT-qPCR all peaked at the second time point in August. Other transcripts including 2 acyltransferases
(comp16498_c0 and comp64068_c0), a desaturase (comp34232_c1), and a keto-ACP synthase
(comp38847_c0) peaked at one or both of the latter time points in September (Figure 7).
The same general gene expression trends were observed between the 2 cultivars for some of the genes we evaluated by qRT-PCR. For example, relative transcript abundance of a hydroxyl-
ACP dehydratase (comp39981_c0), an acyl-activating enzyme (comp40417_c0), and a malonyl-
CoA:ACP transcyclase (comp40800_c0) peaked early in our time course for both cultivars, and
decreased at later time points (Figure 6). In several instances the correlation was not as obvious, and this could be due to primer mismatches due to differences in gene sequence between cultivars, or variations in the timing or selection of gene expressed during kernel development between the cultivars.
Discussion
Past reports have provided a description of embryonic pecan nut development at the morphological and tissue levels, but this area of research has been largely dormant for the past
40 years. This work focused on the third phase of nut development, and characterized the gene expression occurring within a developing pecan kernel. This work is the first of its kind and will provide an opening for extensive related research and development in the pecan industry and lay a foundation for comparing qualitative and quantitative differences among pecan cultivars.
31
Potential changes in the global climate due to increasing carbon dioxide levels could make tree nut (and other crop) production increasingly difficult. The use of the sequence and gene expression information we have generated will allow the comparison of gene expression differences among growth conditions, and defining genetic components that may be used to enhance breeding and identify best growth practices resulting in better harvest and nut quality.
Our work can be used to correlate physiological changes within the developing nut to molecular and genetic markers for the nut developmental timeline, correlate gene expression with plant development and productivity, and characterize the expression and accumulation of pecan allergens.
We have characterized the expression of 3 pecan allergen genes in the Sumner and Desirable
cultivars, identified over 200 fatty acid metabolism genes, and highlighted those that are up- regulated during kernel development. We identify several fatty acid synthesis genes whose expression correlates with the expression patterns of pecan allergen genes. This suggests coordinated regulation of gene expression for these targets during pecan kernel development.
The applied extension of our findings could provide information to identify solutions that may help those who suffer from tree nut allergies and facilitate additional food safety technologies.
32
Acknowledgments
This publication (or project) was supported by the Specialty Crop Block Grant Program at the
U.S. Department of Agriculture through grant agreement number 12-25-B-1464. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the
USDA.
Table 1
Table 1 Total bases assembled 86X106 (bases) max contig length 12160 (bases) average contig length 607 (bases) N50 contig length 873 (bases) total transcripts 142K Unique genes (Trinity) 133K
33
Table 2
C"l11'4lf1' c·n,.·r.,..rt I t•rt'ilk-t..d produf'l Cludrr
s.t "f I xom ttA7-l (:} ..1\l' lu.ao:r1 (lm 17:\ .S .:() 'l\l'lln2 ·om I H '1 . om l:t.sl9 cO l\l$lft' ·om I US5Q cO ' lu ct -1. ••m IW-1. d -.1\l"it.rf-l '.:>ml\1'-104 .:1 ' lll<.tl.'f X'll'll'l7 kJ,tt1 " ...,m.'.Jllf>7 rt1 IUeo:r(i 1Ull l7;'i \.-Q "'"'6 .,,m l-107< ct:t ' tu..t 'lu nl m.,.; t 76 o."() 1\Jsttr$ wnf\J I1f1 lullla •m QQlt7 \,"() "'lu.utor 4 '(onl <)987 dl ' lu!.to:r 10 m J0:!-1'1 cO -;il.J!Il('f JU .,t,il J,t.a1 cO 'lu'il o.'f II '<'m74 cO "tno;ttort l 't'nl 117,\.1 (:(1 'lu.;cd'IZ t>m 11No.i> "'1\hk't 1:: .,,mpi 7Nt c:O ' luqcr 10 :.'m 1&67:! .-.o ':tu!lk'JJU omt: IS{I7, c;1 Tl."l\)("TTl."A'J\JTaAVAUTATr 1Jt'TAVA0VATVA1\NIOMVA , lu,;t..-r t n C.ml'!JQ'Ii90 cO '(U' ' lusao:r to 'OM 961>1 o."' "lutn HI 'Nn JMI c0 "Jt:A.CXX1TMCl' \VN\t'XTITA .'luo,t(fQ 'Nnllf..... ""lwn,C'I'Q :('mr(,(i(..St c{l 'ATf'·VklATf'M1l1,\GGr...AATf' '•"f Hri'•l 'f>m 17\C \F 7-46191 ,,ntr.J :\FI1ol619 1 '-lOAl\JAliA.-\TAAA\.iATAOA ;..Jnlf ol \Fo76174 llntr-61 FU767N T lTJiOlklOATAOALK'.OAt"'Tn..l 34 Figure Legends Figure 1. Cross-section of developing Sumner pecan embryos. Samples were collected based on morphology from individual ‘Sumner’ trees at the Louisiana State University pecan orchard (Shreveport, LA) during August and September of the 2012 growing season. Trees received standard agronomic practices and the nuts are representative of “water” stage (August 11), “gel” stage (August 23), “dough” stage (September 4), and “mature” nuts (September 20). Figure 2. Proximate analysis of developing Sumner pecan embryos. Figure 3. GO annotation of developing Sumner pecan embryo transcriptome. Sequenced and assembled RNA samples from developing Sumner pecan embryos were annotated using the SwissProt, Uniprot, and Uniref90 databases with Trinotate software. Annotated genes were categorized based upon biological process, cellular component, and molecular function definitions. Figure 4. Characterization of significant gene expression changes during Sumner pecan embryo development. HTSeq and DESeq2 were used to count and determine significance of gene expression changes as described in the Trinity abundance estimation and identifying DE feature protocols Figure 5. Pecan allergen gene expression analysis in developing embryos. The gene expression changes of 3 pecan nut allergens, Car i 1 (comp34075_c0), Car i 4 (comp34066_c0), and the 7S 35 vicilin homolog (comp34067_c0) during development were plotted based upon absolute (A) and scaled (B) differences. Figure 6. Clustered gene expression profiles from developing Sumner pecan embryos. Gene expression profiles were evaluated/created in R using kmeans to define 12 unique clusters. Figure 7. Quantitative real-time PCR of select allergen and fatty acid metabolism genes from developing Sumner and Desirable pecan embryos. Gene expression profiles from Sumner and Desirable cultivars were evaluated by RT-qPCR. Four developmental time points are represented. A) Expression of known pecan genes whose products elicit an allergic response in humans. B) Expression of genes involved in various stages of fatty acid biosynthesis. Developmental time points denoted with an asterisk have ≥ 2-fold difference in transcript abundance levels between the two pecan varieties and are significantly different as determined by paired t-test. Figure 1. Cross-section of developing Sumner pecan embryos 36 Figure 2. Proximate analysis of developing Sumner pecan embryos Waiting for proximate analysis figures for each of the time points from Suzi, John, Peter to make table, line graph, or bar chart with sample time and moisture, carbohydrate, protein, and lipid (oleic, linoleic, etc) content Figure 3. GO annotation of developing Sumner pecan embryo transcriptome A 37 B 6000 - 0 4000 - .2 c g c:., 8. 2000 - 38 c lL 4000 - :::;; .e c § 41) 2000 - c: "0>' 0 39 Figure 4. Relative magnitude of gene expression changes during Sumner pecan embryo development A. Transition 1, 8/11-8/23 40 B. Transition 2, 8/23-9/4 41 C. Transition 3, 9/4-9/20 100% 75% 50% 25% 0% 32 100% 75% direction .5.0.% IOWN 25% UP 0% 100% 75% 50% 25% 0% 42 Figure 5. Pecan allergen gene expression analysis in developing embryos A. 43 B. 44 Figure 6. Clustered gene expression profiles from developing Sumner pecan embryos. Figure 6 c: ::> 0 u "0 QJ Iii u V) V) c ::> 8 "0 QJ Iii u V) 45 Figure 7. Quantitative real-time PCR of select allergen and fatty acid metabolism genes from developing Sumner and Desirable pecan embryos. 46 References 1. Pollack, S. a. P., A, Noncitrus Fruits and Nuts 2011 Preliminary Summary. 2012. 2. Blomhoff, R.; Carlsen, M. H.; Andersen, L. F.; Jacobs, D. 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M., Fatty acid profile, tocopherol, squalene and phytosterol content of brazil, pecan, pine, pistachio and cashew nuts. Int J Food Sci Nutr 2006, 57, 219‐28. PROJECT THREE TITLE_- New Landscape Plants for Louisiana SUBGRANTEE: LSU AGCENTER Project Summary Objectives of the project were: (1) Obtain landscape tree and shrub material of genera, species and varieties listed above and establish plants for evaluation in the Piney Woods Garden or the field evaluation planting area at the LSU AgCenter Hammond Research Station. 52 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry (2) Work with growers and retailers to determine interest and potential market of these trees and shrubs. (3) Communicate information to Louisiana’s nursery growers, landscape industry members, retailer garden center managers and employees, and home gardeners/master gardeners on native and exotic landscape trees and shrubs with potential. Home gardening consumers want new and improved ornamental plants for their landscape. One of the focus areas for ornamental horticulture over the years has been the development and selection of new plant cultivars and re-introduction, re-distribution of older existing plant species and cultivars. New plants spark interest among retailers, landscapers, and especially consumers and provide diversity in the landscape. New plants not only spark interest in consumers, but also provide diversity in the landscape. Over- use of a limited numbers of species can be aesthetically boring and biologically irresponsible due to the problems that can develop in a monoculture. The Gulf Coast region is one of the most diverse climates in the United States for selection of plant material from native habitats. The Southeastern region of the United States, which includes the Gulf Coast region, is responsible for up to 50-60% of wholesale production in the country and is also the regional leader in retail sales of ornamental plants. In addition, Louisiana has more nursery growers and plant hobbyists interested in ornamental plant breeding and development (per capita) than most other Southeastern states. This provides a unique opportunity to develop and introduce new species/cultivars to Louisiana’s growers, landscapers, retailers, and gardening consumers. This project will promote the selection and evaluation of novel ornamental plants that have potential for nursery production and landscape use in Louisiana. The emphasis will be on under-utilized native and naturalized ornamental plant species. Through plant evaluation, we can identify suitable plant species and transfer them into the wholesale and retail markets for significant impact on the nursery, landscape and garden center industry. Louisiana has a significant number of hobby, amateur plant breeders/developers who have developed ornamental plants with potential impact upon the nursery and landscape industry. Many of these individuals own wholesale nurseries or are otherwise engaged in the green industry in Louisiana and the surrounding gulf states area and do not have the time and resources for evaluating, promoting, and marketing these new or lesser known plants. They may have these plants in production but sold only small quantities to landscapers and garden centers or directly marketed these plants to home gardeners. 53 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Project Approach Piney Woods Garden We planned, planted, created 34 landscape beds over 5 acres, installed irrigation and pine straw mulch in the new Piney Woods Garden at the Hammond Research Station. The garden is featuring additional plantings of native trees, selections of clonally propagated cypress from China, southern heritage shrubs (such as camellias), native azaleas, a collection of yellow flowering magnolias, Japanese maples, Huang azaleas, new shade tree selections and other plants that are being evaluated for use by nursery and landscape industry. See the accompanying report that includes a current plant inventory of plant species and varieties (400+) established in the Piney Woods Garden at the LSU AgCenter Hammond Research Station. We completed asexual propagation of bald cypress (Taxodium districhum) selections for long term evaluation. We initiated cooperative work with Dr. Mark Windham at the University of Tennessee to conduct evaluation of new dogwood (Cornus florida) varieties in the Piney Woods Garden. We obtained plants via cooperative work with growers / retailers / university partners (see list below) for planting and evaluation studies in the Piney Woods Garden. We initiated a Louisiana iris collection/repository at the Hammond Research Station in the Piney Woods Garden in cooperation with the Society for Louisiana Irises. Additional varieties of Japanese maples were added to plantings at the station in cooperation with Tim and Matt Nichols at Nichols Nursery, East Flat Rock, NC. Field Evaluation Planting Completed establishment of a field production area for long term woody ornamental plant evaluations and selections. This is a 3 acre area featuring “lined out” plantings of crape myrtle varieties for evaluation, Pawlonia trees, Ilex cassine (dahoon holly), vitex, and hardy perennial hibiscus. Planting was initiated April 2013. Field soil preparation work was done, drainage improved and irrigation system installed. Ten Confederate rose (Hibiscus mutabilis) varieties and selections are currently being greenhouse grown and will be planted in spring 2014. An additional 50 selections (unnamed varieties) of hardy perennial hibiscus are in the greenhouse and will be planted in out-field studies for selection purposes in spring 2014. Worked with Walter’s Garden, Fleming Flower Fields, Color Spot Nursery, Mississippi State University, and the Louisiana Society for Horticultural Research to establish a long term field studies on adaptability and landscape performance. Varieties planted June 2013 with data taken twice monthly through early October. Study continues in 2014 and 2015. 54 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Initiated long term evaluation of 150 parsley hawthorn selections in cooperation with horticulture professor Charlie Johnson and post-doctoral researcher Daniel Wells. Initiated plant breeding efforts on Vitex and completed a planting of white flowering seedlings for future selection. Conducted asexual propagation studies on ten named Vitex varieties. A landscape trial including most of the recently introduced crape myrtle cultivars has been initiated in the field planting since April 2013. These collections include the Early Bird, Razzle Dazzle, Enduring Summer, Delta, Barnyard, Magic and Ebony (aka Black Diamond ) series in addition to several other cultivars. The Early Bird series has been on the market the longest and was introduced by Plant Development Services, Inc. These cultivars are part of the Southern Living Plant Collection. These are dwarf growing plants maturing at 4 feet. Early Bird Lavender (soft lavender) is promoted as a very heavy earlier bloomer. Other cultivars include Early Bird Purple and Early Bird White. For several years, the burgundy foliaged Delta Jazz (semi-dwarf, brilliant pink) from Plant Development Services, Inc. has been a stand-alone cultivar. This cultivar is also part of the Southern Living Plant Collection. Five year old plants of Delta Jazz are 8 feet tall in most locations. Plants are classified as semi-dwarf growers, which normally indicates heights ranging from 8-12 feet. New for 2014 are four new color additions – Delta Breeze (light lavender), Delta Eclipse (brilliant purple), Delta Ebony (white) and Delta Flame (dark red) The new dark burgundy foliaged Ebony series from Cecil Pounders at the USDA-ARS are also being sold under the Black Diamond name by J. Berry Nursery. These plants mature at 8 feet and retain foliage color spring through fall. Ebony Cultivar Name Black Diamond Cultivar Name Ebony and Ivory (white) Pure White Ebony Flame (dark red) Best Red Ebony Embers (deep red) Red Hot Ebony Fire (dark red) Crimson Red Ebony Glow (light pink to white) Blush Red Rooster (brilliant red), Purple Cow (deep purple) and Pink Pig (soft pale pink) are being sold as “mid-sized” growers and are promoted collectively as the “Barnyard Favorites” in the Gardener’s Confidence Collection. Red Rooster is “something to crow about”; Pink Pig is something to “squeal with delight” and Purple Cow can be used to create an “udderly majestic garden.” Also in the Gardener’s Confidence Collection is the Razzle Dazzle crape myrtles. These have been around for 8 years or so now. True dwarf habits at 4 feet, cultivars are Berry 55 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Dazzle (fuchsia), Cherry Dazzle (cherry red), Dazzle Me Pink (pink), Diamond Dazzle (pure white), Strawberry Dazzle (neon rose) and Sweetheart Dazzle (pink). Cherry Dazzle has been a longtime exceptional performer in LSU AgCenter landscape trials. The Magic series from Plant Introductions that are now part of the First Editions program by Bailey Nurseries includes Coral Magic (salmon pink), Purple Magic (dark purple), Plum Magic (fuchsia pink), Moonlight Magic (white) and Midnight Magic (dark pink). Coral Magic and Purple Magic have reddish new foliage in the spring. Plum Magic has plum purple foliage in the spring. Midnight Magic has purple maroon foliage that persists from spring to fall and Moonlight Magic has dark maroon foliage that persists from spring to fall. These are semi-dwarf plants (6-10 feet tall at maturity for Coral, Purple and Plum, while Midnight matures at 4-6 feet and Moonlight matures at 8-12 feet) and were developed by Mike Dirr in Georgia at Plant Introductions, Inc. The Princess series is a new dwarf group developed by Dow Whiting at Garden Adventures Nursery in Missouri and is being marketed as part of the Garden Debut program by Greenleaf Nursery. This series includes Holly Ann (cherry red), Kylie (magenta pink), Zoey (cherry red with cotton candy pink) and Lyla (rose pink). Ball Ornamentals has the new Enduring Summer collection of crape myrtles. Cultivars are Enduring Summer Red, Enduring Summer Fuchsia, Enduring Summer Pink, Enduring Summer White and Enduring Summer Lavender. These plants were also developed by Mike Dirr. These are reported to have re-blooming characteristics. The Enduring Summer cultivars have an upright habit and mature height is 5-6 feet with a 4- foot spread. At the LSU AgCenter, we also have plants of Bayou View which is the Lagerstreomia fauriei national champion single trunk crape myrtle tree located at Akin’s Nursery in Shreveport. We are also evaluating several lavender flower crape myrtles in cooperation with John Davy at Panhandle Growers in Florida. Work with Growers / Retailers and University Cooperators Worked with Moran’s Nursery in Baton Rouge to obtain Nova pentas (lost to commerce cultivar) for propagation and distribution at the Louisiana Society for Horticulture Research annual conference in Lafayette, LA in March 2013. Propagated Florida Dwarf Rose purslane (lost to commerce cultivar) for propagation and distribution at the Louisiana Society for Horticulture Research annual conference in Lafayette, LA in March 2013. Obtained three heirloom verbenas from former LSU AgCenter horticulturist Carlos Smith’s collection and initiated cutting propagation to increase stock. 56 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Obtained woody ornamental plants from David Creech at Stephen F. Austin State University for propagation and evaluation. Traveled to Doyline, LA to visit with Bud Willis at Willis Farms to obtain new cultivars of Ilex decidua for performance characteristic evaluation. Initiated securing 25 advanced selections of Ilex cassine from Buddy Lee at Transcend Nursery in Independence, LA for long term landscape performance characteristic and selection evaluation. Increased collection of turk’s cap species and cultivars in cooperation with Jeff McMillian at Almost Eden Plants in Merryville, LA. Traveled to McMinnville, TX to secure plant material (dogwoods, new hydrangeas and yellow flowering magnolias) from USDA-ARS horticulturist Donna Fare. Traveled to Mobile, AL to obtain plants from Plant Development Services, Inc. – Southern Living camellia collection and heat tolerant rhododendrons for landscape performance characteristic evaluations. Traveled to Stephen F. Austin State University to exchange plants with Dr. David Creech. Nursery and university cooperators for this project have included: Clegg’s Nursery, Baton Rouge, LA Almost Eden Plants, Merryville, LA Plant Development Services Inc., Loxley, AL Green Nurseries and Landscape, Fairhope, AL GreenForest Nursery, Perkinston, MS USDA-ARS, Poplarville, MS USDA-ARS, McMinnville, TN Garden Design Nursery, Danielsville, GA Nichols Nursery, East Flat Rock, NC Transcend Nursery, Independence, LA J Berry Nursery, Grand Saline, TX Panhandle Growers, Milton, FL Stokely Nursery, Semmes, AL SFA State University, Nacogdoches, TX Willis Farms, Doyline, LA Louisiana Growers, Amite, LA Jenkins Farm and Nursery, Amite, LA Bracy’s Nursery, Amite, LA Windmill Nursery of Louisiana, Folsom, LA Greenleaf Nursery, El Campo, TX Ingram and Sons Nursery, Homestead, FL 57 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Auburn Univ. Ornamental Station, Mobile, AL Durio Nursery, Opelousas, LA Arcola Nursery, Roseland, LA TreeSearch Farms, Houston, TX Moran’s Nursery, Baton Rouge, LA University of Tennessee, Knoxville, TN Mississippi State University, Starkville, MS Goals and Outcomes Achieved Planned and held the inaugural Margie Jenkins Azalea Garden Horticulture Lecture Series on May 17, 2013, to highlight new plant development highlights and information on plant selection based on native geographical adaptability. Guest speakers were David Creech from Stephen F. Austin State University and Geoff Denny from Mississippi State University. Event also included the debut of the Piney Woods Garden planting. 75 nursery and landscape professionals attended the event (objective 1). Planned and held the 2013 Master Gardener Appreciation Day for May 10, 2013, with plant distribution included and showing of research plots containing new landscape plant research (objective 2 and 3). Horticulture faculty member Allen Owings and others presented from October 2013- September 2013 thirty six educational programs to nursery and landscape professionals and forty three educational programs to home gardeners/master gardeners highlighting new plants being evaluated at the LSU AgCenter’s Hammond Research Station (objective 2 and 3). Horticulture professor Allen Owings gave a presentation to attendees at the Southern Plant Materials Symposium during the SERA 27 Nursery Crop and Landscape Systems annual meeting in Fletcher, NC in June 2013 highlighting field evaluation trial efforts at the LSU AgCenter Hammond Research Station. 50 green industry professionals and ten horticulture faculty colleagues from the SE USA attended (objective 3). Participated in Louisiana Society for Horticulture Research activities and worked with members on plant exchange and future plant distribution efforts (objective 2 and 3). Horticulture professors Allen Owings and Ed Bush attended the annual meeting of the Southern Region – International Plant Propagator’s Society in Auburn, AL. This meeting provided educational sessions on new plant materials for the SE and included a plant exchange and auction (objective 2 and 3). 58 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Presented new landscape plant information at the Gulf States Horticultural Expo in Mobile, AL (objective 3). Attended Louisiana State Horticulture Society annual meeting in Lake Charles, LA and distributed and obtained plants with landscape potential currently not utilized or underutilized in Louisiana (objective 3). Planned the 2013 Landscape Horticulture Field Day at the LSU AgCenter Hammond Research Station for October (objective 2 and 3). Published and distributed via e-mail 26 editions of the LSU AgCenter Trial Garden Report from the Hammond Research Station highlighting new ornamental landscape plants being evaluated at the LSU AgCenter Hammond Research Station (distribution 950) (objective 3). Published and distributed via e-mail 34 LSU AgCenter Ornamental Horticulture E-News Updates from the Hammond Research Station that highlights educational programs, events, plant trials and additional ornamental horticulture efforts (distribution 950) (objective 3). Planned the Louisiana Plant Materials Conference for master gardeners in November 2013 and the Louisiana Plant Materials Conference for green industry professionals in December 2013. Both events will include presentations on plant trials at the LSU AgCenter Hammond Research Station that was initiated with funding from this project (new crape myrtle cultivar evaluations, three year hardy hibiscus landscape study, and new plants in the Piney Woods garden) (objective 3). Attended Southern Nursery Association Research Conference in Atlanta to provide a presentation on new plant development at the LSU AgCenter (objective 3). Attended the Southern Plant Conference in Atlanta. Received an invitation to provide a presentation at the Southern Plant Conference in July 2014 to include information on plants from the new Piney Woods Garden at the LSU AgCenter Hammond Research Station (objective 3) Provided information on new plant development at the LSU AgCenter at the AgCenter booth and during an oral presentation at the Nursery and Landscape Expo in Dallas, TX in August 2013 (objective 3). Beneficiaries Measurable outcomes of this project were to collect and develop a list of possible new plants for the ornamental horticulture industry. 400-450 plant varieties were added to the 59 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Piney Woods Garden and Field Evaluation Planting at the LSU AgCenter Hammond Research Station for evaluation purposes since the initiation of this project. Surveys of wholesalers and retailers were to be used to monitor acceptance of plants. Our goal was to increase use of new and under-utilized ornamental plants by nursery and landscape professionals. At events held from late fall 2012 through mid-spring 2013 at the LSU AgCenter Hammond Research Station, program participants (included nursery growers, retailer garden center managers and employees, landscape horticulturists and master gardeners) were surveyed for their preference for specific plant evaluations in several general categories. The results: Native Trees and Shrubs – Improved Varieties 26.6% Louisiana Super Plants 23.2% Fruit Species/Varieties for Landscape Use 21.7% Japanese Maples –Variety Selections for the South 18.7% Alternative Warm-season Bedding Plants for Shade 18.2% Roses – Low-care Varieties 17.7% Alternative Cool-season Bedding Plants 15.3% Hardy Hibiscus and Other Unique Hibiscus Species 13.8% Tropical and Semi-Tropical Plants 13.3% Azaleas – New Multi-Seasonal Bloomers 12.8% At two events held at the LSU AgCenter Hammond Research Station in fall 2013 (study conclusion), a similar mix of program participants were asked about these categories and there was increased interest in Japanese maples (increase from 18.7% to 33%), improved varieties of native trees and shrubs (increase from 26.6% to 45%), hardy hibiscus and other hibiscus species (increase from 13.8% to 30%), and Louisiana Super Plants (increase from 23.2% to 33%) as a result of ornamental plants being added to research efforts at the Hammond Research Station. Other plant categories showed results similar to the initial survey. In the concluding surveys, 100% of nursery growers, retailers and landscape horticulturists surveyed viewed/observed plants in the Piney Woods Garden and/or Field Evaluation Area that they planned to add, use, produce, or sell in their business. This response was equivalent to 25-35% of the plants viewed/observed by retailers. Landscapers surveyed desired to begin planting/using/selling around 35% of the plants viewed/observed. Growers desired to initiate production of 10-12% of the plants viewed/observed. Our target was to expect a 10% increase in use of the introduced ornamental plants by the industry. In addition to the plant categories included in the survey, participants expressed interest in these specific plants for possible marketing, promotion, Super Plant designation 60 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry efforts, etc. in the future – Twist of Pink oleander, Chinese fringe tree, dogwood, altheas, hardy hibiscus, various cypress species, dwarf camellias, vitex and abelias. Groups benefiting from this project include nursery growers in Louisiana (approximately 600), landscape horticulturists (2200) and independent retail garden centers (500). Nursery growers’ wholesale farm gate value annually in Louisiana is approximately $130 million. Landscape horticulturists and similar business generate $800 million to the Louisiana economy annually with another $200-300 million generated by independent retailers. Lessons Learned This project has resulted in increased awareness of landscape horticulture research and extension activities at the LSU AgCenter Hammond Research Station, especially among nursery growers (in state and out of state), landscape horticulturists and retail garden center owners, managers and employees. We experienced no unexpected outcomes or results that affected project implementation. Goals and outcome measures were achieved. Retailers, landscapers and growers have different perceptions of landscape plant introductions. Future projects need to address the need to encourage growers to better work with retailers and landscapers (on maybe a contract basis) to produce plants that they fear will not sell due to lack of clientele acceptance. Additional Information News Article – January 2013 www.lsuagcenter.com/news_archive/2013/january/headline_news/hammond-research- station-serves-louisiana-landscape-industry-.htm News Article – June 2013 www.lsuagcenter.com/news_archive/2013/june/headline_news/research-station-visitors- vote-on-favorite-plants-.htm News Article – September 2013 www.lsuagcenter.com/news_archive/2013/september/headline_news/landscape- horticulture-field-day-scheduled-for-oct-10-in-hammond.htm LSU AgCenter Ornamental Horticulture Updates Posted on LA Nursery and Landscape Association’s Website with Information That Includes Results of Work from this Grant Project Conducted at the Hammond Research Station www.lnla.org/CurrentNews/OrnamentalHorticultureUpdate/tabid/81/Default.aspx LSU AgCenter Trial Garden Reports Updates Posted on LA Nursery and Landscape Association’s Website with Information That Includes Results of Work from this Grant Project Conducted at the Hammond Research Station www.lnla.org/CurrentNews/TrialGardenReport/tabid/126/Default.aspx 61 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Plant Listing for New Piney Woods Garden at the LSU AgCenter Hammond Research Station is available in Excel format if requested. One of the 60 horticulture tour groups have seen research from this project endeavor at the LSU AgCenter Hammond Research Station. New Field Evaluation Area 62 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Gordonia – Piney Woods Garden Hardy Hibiscus Field Planting Yellow Magnolias – Piney Woods 63 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Rare Neches River Mallow New Althea to Be Released Contact Person Allen Owings Professor (Horticulture), LSU AgCenter, Hammond Research Station 985.543.4125 [email protected] 64 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry PROJECT FOUR TITLE_- Increasing Competitiveness of Louisiana Specialty Crop Producers with MarketMaker SUBGRANTEE : LSU AGCENTER Project Summary • Provide a background for the initial purpose of the project, which includes the specific issue, problem, or need that was addressed by this project. The problem for Louisiana’s specialty crop growers is that there may not be enough existing, new, or alternative market outlets to encourage increases in production or new ventures into alternative or non-traditional crops. Additional tools are needed to find markets for local products. Electronic search tools such as MarketMaker have become one answer to these marketing issues. MarketMaker enhanced the competitiveness of Louisiana’s specialty crop growers through easier and broader identification of buyers. The Louisiana MarketMaker program provided an easily accessible point of contact for consumers seeking locally grown products. For this reason, we wanted to focus outreach and recruitment efforts to Louisiana specialty crop growers with this project. By targeting specialty crop growers and the venues at which they met and gathered information (producer or industry meetings, trade shows, etc.), we expected to increase awareness of the benefits of Louisiana MarketMaker to specialty crop growers, expand marketing opportunities for these growers, and to increase awareness and use of Louisiana MarketMaker by the buyers of specialty crops (consumers, wholesale and retail food buyers, restaurants, grocers, etc.). • Establish the motivation for this project by presenting the importance and timeliness of the project. ‘Locally produced’ is a very popular food concept, as documented in the popular press and academic literature. It offers the opportunity to gain customers by differentiating on many levels such as a product being produced locally, and to build additional levels of differentiation based on other product attributes. Specialty crop growers in many states, including Louisiana, have taken advantage of this preference, particularly through MarketMaker. With this tool, Louisiana’s specialty crop growers have become more competitive through expanded markets and production efficiencies from larger activity levels. Better markets lead to stronger farm and rural communities. Many growers lack strategic marketing skills and time necessary to devote to finding markets. With this project, it was important to support and improve marketing efforts of small and medium sized specialty crop farming operations by providing a technology to identify new and alternative markets through a professionally developed Internet format like MarketMaker. When such internet-based market opportunities, have outreach and 65 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry education efforts targeted to specialty crop growers and buyers it increases specialty crop availability directly to consumers, to restaurants, to other foodservice outlets. The MarketMaker tool also enables the commercial trade (restaurants, food buyers and traditional grocers, large and small) to identify new sources of specialty crops. The larger objective is to support specialty crop sector of agriculture (both growers and buyers); to enable consumers to have access to healthy fruits and vegetables and to other specialty crop products and producers to increase marketing opportunities and profit potential through a program like Louisiana MarketMaker. • If the project built on a previously funded project with the SCBGP or SCBGP-FB describe how this project complimented and enhanced previously completed work. No previous funding for this project from SCBGP or SCBGP-FB was received. Project Approach • Briefly summarize activities performed and tasks performed during the grant period. Whenever possible, describe the work accomplished in both quantitative and qualitative terms. Include the significant results, accomplishments, conclusions and recommendations. Include favorable or unusual developments. Over this reporting period (10/1/2012-9/30/2014), we have conducted the following activities toward achieving our goals. Supporting materials (push cards) for specialty crop growers that we developed earlier in the project, we disseminated more than 20,000 push cards to potential users of the Louisiana MarketMaker program that provided information on registering a food business or using the Louisiana MarketMaker website to locate businesses, particularly farmers growing specialty crops. We gave more than 120 presentations on Louisiana MarketMaker program, focused on how to register online, as a specialty crop grower or a food business focused on specialty crops or how to use the Louisiana MarketMaker website to locate and buy from specialty crop growers and food business specializing in specialty crops. These efforts contacted more than 1,650 specialty crop growers in Louisiana as well as 15,000 people who buy or use specialty crops in Louisiana. The Extension Associate, Mr. Cooper, was supported in large part by resources from this project. All of his efforts supported by this project were directed and targeted exclusively to specialty crop producers and buyers. Using resources of this project, the Extension Associate and PIs participate in meetings that were targeted to either buyers of specialty crops (thus enhancing potential marketing opportunities for specialty crop producers) or events that focus on growers of specialty crops. Any efforts for Louisiana MarketMaker directed toward other commodities were performed by other personnel and/or used 66 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry funding from other sources. At no time during this grant period did the Extension Associate present at anything other than these types of shows. We administered two surveys – one to specialty crop producers and the other to consumers or buyers of specialty crops. Both surveys were administered electronically via email. For the producer survey, designed to evaluate MarketMaker as a direct marketing tool for specialty crop buyers, we surveyed approximately 460 specialty crop growers, whose names we gathered from the various specialty crop growers associations in Louisiana by, among others, Louisiana Vegetable Growers Association, LDAF, Louisiana Mayhaw Association, etc. Of these, 133 responded with usable information. Respondents indicated that direct-to-consumer selling was the most common marketing practice, with roadside stands and pick-your-own being used by 50% of growers. Though over 50% used the internet for farm business, only 40% of those growers who used the internet used it for selling specialty crop produce or products. And of those growers sold produce online, less than 10% were aware of Louisiana MarketMaker. Average sales of growers, across all marketing channels was less than $50,000 annually. The email survey to consumers, randomly selected from lists of residents from Louisiana Public Policy Research Lab (at LSU), were administered to 1,000 individuals. Usable response were obtained by 115. Of these respondents, less than 5% were aware of the Louisiana MarketMaker website or program. Many respondents (34%) purchased specialty crops at farmers markets, roadside stands or pick-your-own farmsteads. Very few purchased specialty crops online (11%) and even fewer had used MarketMaker for locating and purchasing specialty crops (3%). Nonetheless, of those who had used MarketMaker, a large majority (78%) were satisfied with MarketMaker and would use it again to locate specialty crops in Louisiana. These efforts resulted in 102 additional registrations of specialty crop producers in Louisiana on the Louisiana MarketMaker website during the reporting period. Additionally, there was an increase of 5-10% in monthly website visits and unique visitors during the reporting period. We concluded from these findings that additional resources deployed in marketing this online marketing program to specialty crop producers would be beneficial to their business as it increases awareness and web traffic for specialty crop growers and buyers of specialty crop produce. • Present the significant contributions and role of project partners in the project. None to report. Goals and Outcomes Achieved • Supply the activities that were completed in order to achieve the performance goals and measurable outcomes for the project. 67 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry We gave more than 120 presentations on Louisiana MarketMaker program, focused on how to register online, as a specialty crop grower or a food business focused on specialty crops or how to use the Louisiana MarketMaker website to locate and buy from specialty crop growers and food business specializing in specialty crops. These efforts contacted more than 650 specialty crop growers in Louisiana as well as 15,000 people who buy or use specialty crops in Louisiana. Presentations were given, several times each, to: 1. Baton Rouge Food Policy Council in the greater Baton Rouge area; 2. Baton Rouge Farm to School program, making contact with potential buyers (within EBR Public School System and Diocese of Baton Rouge Parochial School System) of local specialty crops food products and specialty crop farmers; 3. Louisiana Restaurant Association (LRA) meeting and Farm to Table symposium in New Orleans – a meeting at which we presented, including specialty crop farmers and agribusiness leaders representing firms that purchase and were seeking more opportunities for procuring Louisiana-grown produce and food products made from specialty crops for their firms; 4. Annie’s Project – meetings targeting female specialty crop producers or female- owned business seeking to purchase from specialty crop farmers; 5. Farm to School meeting in Alexandria; 6. Louisiana Ag Expo in Monroe at which we contacted more than 100 Louisiana specialty crop growers; 7. Louisiana Food Processors Conference in Baton Rouge at which we made contact with 150 specialty crop producers and food products buyers and processors; 8. Southern University Small Famer’s Conference at which we contacted more than 75 specialty crop growers, most of whom were minority-run farming operations; 9. Coushatta Ag Conference, focused on Native Americans involved in specialty crop farming and interested in starting a farmers market; made contacts with over 500 individuals at this event; 10. Connect My Louisiana (CML) program, delivered throughout the state targets rural households in economic opportunities with electronic commerce, using programs like Louisiana MarketMaker; and 11. Farm Bureau Convention in New Orleans at which we contacted more than 100 specialty crop producers; 12. Louisiana Feast of Fields event in Baton Rouge at which we made contact with 25 buyers and processors of fruits and vegetables and 10 specialty crop farmers; 13. Farm Bureau Ag Labor Seminar (topic keenly important to specialty crop farmers) in Baton Rouge at which we contacted more than 100 commercial specialty crop famers; 14. Choctaw Indian Tribe at which we contacted 10 specialty crop famers; all of whom were Native American farmers; 15. Strengthened relationship with several area Farmer’s Market (Lafayette, Baton Rouge Red Stick, and New Orleans Crescent City) so that the Louisiana MarketMaker Program could more closely and efficiently help commercial specialty crop producers and buyers via this farmer’s market 68 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Using resources of this project, our Extension Associate targeted buyers of specialty crops (thus enhancing potential marketing opportunities for specialty crop producers) or growers of specialty crops. Now it is obvious we cannot limit participation on the MarketMaker website to only specialty crop producers or buyers, but all of our activities supported by this project were targeted solely to specialty crops. Efforts directed at other commodities were supported by other funding sources and carried out by other personnel. It is also obvious we cannot limit participation at displays or at meetings to only specialty crop producers or buyers; we greeted any producer or buyer who walked up and started talking with us about MarketMaker. If they needed further assistance they were referred to other Market Maker staff. However, all of our activities supported by this project were targeted directly to specialty crop producers or buyers. • If outcome measures were long term, summarize the progress that has been made towards achievement. Outcomes were short-term and reported below. • Provide a comparison of actual accomplishments with the goals established for the reporting period. See description below. • Note significant contributions or role of project partners (industry groups) in the project during this period. Role of industry groups are included below in description. Primarily hosted events at which we participated and presented on Louisiana MarketMaker program. • Clearly convey completion of achieving outcomes by illustrating baseline data that has been gathered to date and showing the progress toward achieving set targets. All data included in metrics described below each goal of the project. Goal 1: Louisiana MarketMaker will exceed the number of website hits, unique visitors and registered farm business on the Mississippi MarketMaker website by 10% by end date. While MarketMaker is in place in 20 states, Mississippi’s site went online late in 2008 – two years prior to Louisiana. As a neighboring state, it provides a reasonable standard or benchmark over an equivalent startup period of implementation. • Our first goal was to generate more “hits”, unique visitors, and registered business on the Louisiana MarketMaker website by the end of this project than Mississippi MarketMaker. To document and monitor performance toward meeting the benchmark and target, we collected data in normal operations by Louisiana MarketMaker and 69 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry National MarketMaker since the Louisiana MarketMaker program went “live” online in September 2010. • Over the reporting period, as can be seen in the chart on the next page, the Louisiana MarketMaker website has received an average of approximately 98,000 “hits” or visits each month. Mississippi MarketMaker, on the other hand, has received approximately 69,000 “hits” per month since October, 2012. This translates into 42% more “hits” for the Louisiana site than for the Mississippi site; thus meeting our first goal for the project. • Cumulative visits to the site since October 1, 2012 for Louisiana MarketMaker were 2,350,628 and for Mississippi MarketMaker were 1,650,354. Again Louisiana’s site had 42% more visits than Mississippi. See graph below. • Over that same time period, since October 1, 2012, the number of unique users for the Louisiana MarketMaker site averaged 10,170 while Mississippi MarketMaker site averaged 4,999. Louisiana’s site had 103% more unique users as Mississippi MarketMaker. Again, meeting and exceeding our benchmark for this goal. • This is important because the more unique users visiting the site, this increases the probability that they will contact a specialty crop farmer, or any other business on the site, and buy or sell food or food products. See graph below. • As of the end of the project, 102 new, unique user profiles (registered businesses) had been created by specialty crop farmers in Louisiana. By contrast, during that same time period, Mississippi MarketMaker registered 50 new specialty crop farmers to their site. • Thus we met the goal of exceeding the benchmark of Mississippi MarketMaker by 10% in terms of website “hits”, unique visitors to MarketMaker, and new specialty crop growers registering their business on MarketMaker. Goal 2: Louisiana MarketMaker will increase grower awareness of MarketMaker as an aid in direct marketing; 10% greater than comparable states by end date. • As a benchmark, we believed Louisiana’s grower would use Louisiana MarketMaker as a direct marketing tool at a rate 10% greater than those of comparable states, such as Mississippi. To document and monitor performance toward meeting benchmark and target, we conducted a survey of specialty crop buyers and growers to gauge awareness and use levels, and problems or issues. To accomplish this goal, we surveyed specialty crop producers and buyers (consumers), to gauge awareness of and use of direct marketing opportunities like MarketMaker. Our analysis of the survey 70 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry responses indicated that specialty crop growers were much more aware of the Louisiana MarketMaker program in 2013, after we had started this Specialty Crop supported initiative. Evidence of this increased awareness is demonstrated by more than 100 specialty crop producers registering on the Louisiana MarketMaker website during the reporting period while Mississippi MarketMaker had 50 new specialty crop growers registered. However, we were unable to discover that those growers used direct marketing 10% more than previously. We were only able to determine that more specialty crop growers were registered and using MarketMaker; not the intensity of that use. Beneficiaries • Provide a description of the groups and other operations that benefited from the completion of this project’s accomplishments. Beneficiaries included growers and marketers of specialty crops – vegetables and fruits, tree nuts, herbs, greenhouse crops, and others. The ‘food trade’ including foodservice companies such as Associated Grocers; wholesalers such as Capitol City Produce; local and national (Whole Foods, other chains) retailers; institutional servers such as LSU Food Service; and farmers’ markets - all of whom with which we interacted during our presentations and workshops described above. As indicated above, we had direct contact with 650 specialty crop growers in Louisiana at trade shows, producer meetings, food expos, etc. Additionally, we contact 460 specialty crop producers via our email survey and questioned them about MarketMaker. The producer survey funded by this project was directed exclusively toward specialty crop growers or buyers of specialty crops. Overall contact with specialty crop growers exceeded 1,100. Institutions such as Farm Bureau, La. Department of Agriculture and Forestry, and organizations such as La. Vegetable Growers Association, La. Strawberry Commission, La. Sweet Potato Commission and economic development associations were able to better serve clientele because their clients (specialty crop producers) were better able to access markets for their produce as a result of increased traffic afforded them by registering their business on Louisiana MarketMaker. These commissions and associations, and others with whom we had direct contact, represent more than 14,000 specialty crop growers in Louisiana with gross sales of produce exceeding $142 million in 2013. Consumers were able to benefit from increased access to fresh, local produce raised by Louisiana specialty crop producers. More than 15,000 consumers were contacted directly at various trade shows, food expos, farmers markets, producer meetings and other venues that showcased local food, particularly local specialty crop produce and products. 71 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry • Clearly state the quantitative data that concerns the beneficiaries affected by the project’s accomplishments and/or the potential economic impact of the project. MarketMaker provides growers of specialty crops access to more varied and different markets. The number of potential buyers is larger with the advent of MarketMaker. With this access, some preferred products of vendors sell at higher prices, and sales have expanded. The program reduces the need for growers to spend time searching for markets and allows that resource to be reallocated to more profitable alternatives, like production and marketing. One measure of usefulness of the MarketMaker program is the number of visitors to the website. As indicated below in the first chart (1), over the past four years monthly website visits or “hits” to the Louisiana MarketMaker site have averaged over 102,000. This was approximately the same traffic before and after this Specialty Crop Initiative. However, accompanying the website “hits” indicating the number of unique visitors seen in the second chart (2) below. This is a more important indicator of average number of unique buyers on the website. As can be seen, the number of unique visitors (potential and actual buyers and sellers) has increased by 42% since October 2012 – the start of this project focused on specialty crops in Louisiana. The increase, from 7,100 to 10,200 is an increase of over 3,000 per month in the most recent period. The data indicate that more users of MarketMaker are accessing the site, and that number has been trending upward for over the past two years. In fact, of the 20 states currently participating in the national MarketMaker effort, Louisiana ranks third among them and alone accounts for 10 percent of total traffic website nationwide. 72 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry 73 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Lessons Learned • Offer insights into the lessons learned by the project staff as a result of completing this project. This section is meant to illustrate the positive and negative results and conclusions for the project. A few lessons learned from this project are the following. First, outreach effort to specialty crop producers should focus first on meetings of their respective associations for each crop, such as strawberries, blueberries, pecans, sweet potatoes, etc. They are much more likely to be attending these meetings and be receptive to a speaker talking about a new program like MarketMaker, than if they just happen to be at a producer meeting in their country or Parish. Our biggest impact on registration – getting producers to create a profile or register on MarketMaker website, occurred when we had a laptop computer with us so that they could register “then and there”. Field days for specialty crops was a good way to educate people about MarketMaker, but not good for signing them up as they were too busy or too distracted by other field day events to want to register on the laptop be brought along. Industry trade shows, like the Louisiana Restaurant Association meeting, was a great opportunity to contact potential buyers of specialty crops and alert them to the benefits of using MarketMaker to procure such produce. We also successfully used special events like “Feast in the Fields” to showcase locally produced produce by growers registered on MarketMaker and thus demonstrate the type and quality of locally available produce found on the MarketMaker website. This helped generate awareness and business for these growers, and hopefully others registered online growing similar specialty crops. The outreach and extension efforts to the specialty crop growers resulted in significant increase in access to online marketing of specialty crop produce for the growers who used the Louisiana MarketMaker website. First and foremost is the increase of more than 100 specialty crop producers in the database of registered producers on the Louisiana MarketMaker website. Second, the substantial increase in the number of unique visitors or users of the website, demonstrated in the graph (2) above, clearly shows the value of continued outreach efforts. Marketing the Louisiana MarketMaker program to specialty crop producers, and the buyers of that produce are critical to the continued expansion of the specialty crop market in Louisiana. Though not all the website traffic is solely targeted to specialty crops, the vast majority of registered producers on the website are specialty crop growers. Therefore, it stands to reason that increased website traffic is associated in large part to the increased number of specialty crop producers registered and using the Louisiana MarketMaker website. • Provide unexpected outcomes or results that were an effect of implementing this project. 74 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry No unexpected outcomes were realized. • If goals or outcome measures were not achieved, identify and share the lessons learned to help others expedite problem-solving. All goals and metrics proposed in the project were achieved. Contact Person Dr. John Westra, Professor, Department of Agricultural Economics and Agribusiness, Louisiana State University AgCenter, Baton Rouge LA Telephone Number: 225-578-2721 Email Address: [email protected] Additional Information Informational Cards Front Back 75 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry PROJECT FIVE TITLE_- Understanding Asian Citrus Psyllid and Candidatus Liberibacter Asiaticus Population Genetics in Louisiana SUBGRANTEE : LSU AGCENTER Project Summary Project Purpose The confirmation of “Liberibacter-positive” citrus trees in Texas and California in 2012 increased the level of concern the industry has about their future. It also increased concern from other citrus growing states that have not dealt with Huanglongbing (HLB). While the Asian citrus psyllid, Diaphorina citri (ACP) has been established in the south- central US for several years, this was the pathogen not detected in Louisiana and Texas until 2008 and 2010, respectively. Based on what we know about this patho-system, Liberibacter must have entered the state through root stock (not likely because this was an older, established tree) or through the invasion of infective insects. From here, expansion of the disease will occur in one of two ways: 1.) through secondary spread from this infected tree or the insects that have been in contact with it or 2.) through additional primary infections that come from similar points of origin. Therefore, knowing where this infection originated is of paramount importance to reducing future risk to the industry. Timeliness of the Project The ACP samples collected in 2013 from Louisiana will provide information on the population origin and the greening outbreak occurred in 2012. We collaborated with Dr. Blake Bextine (University of Texas at Tyler) to compare the population of ACP from different geographical regions (although funding for Texas was not approved, but we shared the DNA from tissue and ACP). 76 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Project Approach Activities Performed • Mr. Timothy Burks (extension associate hired for this project) attended the 3rd International Research Conference on Huanglongbing-IRCHLB III, Orlando, Florida. February 4-8, 2013. • Adult Asian citrus psyllids were collected from Belle Chasse, LaRose and New Orleans and Houma. • Asian citrus psyllids were not found in Baton Rouge and Hammond. • Pre Survey about Citrus Greening and its Vector Asian Citrus Psyllid. • Total genomic DNA from ACP specimens collected from Belle Chasse, LaRose, New Orleans and Houma sites was extracted according to work instructions W1- B-T-1-16. • Total genomic DNA from suspected citrus tissue samples collected from Belle Chasse, LaRose, New Orleans and Houma sites was extracted according to work instructions W1-B-T-1-17. • All tissue and ACP DNA samples were tested for presence of Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus using a conventional PCR. All ACP and tissue DNA were negative for both strains of citrus greening. • DNA from all ACP samples was shared with Dr. Bextine at University of Texas in Tyler for population studies. • Post survey was sent out to 35 citrus growers in Louisiana and 29 growers responded to the survey. • Meeting with citrus growers was conducted on March 11, 2014 to discuss prevention and management of citrus greening. 77 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry • Dr. Bextine is now analyzing the phylogenetic relationship of different ACP population Goals and Outcomes Achieved • Population studies of Asian Citrus Psyllid Total genomic DNA from ACP specimens and citrus tissue collected from Belle Chasse, LaRose, New Orleans and Houma sites was extracted according to work instructions W1- B-T-1-16 and W1-B-T-1-17, respectively. All ACP and tissue DNA were negative for both strains of citrus greening and DNA from all ACP samples was shared with Dr. Bextine at University of Texas in Tyler for population studies. Preliminary results have revealed that the Asian Citrus Psyllid populations from Louisiana, Florida and Texas share the same genetic origin and the ACP population from Mexico has a different genetic background (Figure 1). 78 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Figure 1: Aligned consensus 18S sequences from Drosophila melanogaster and studied Asian Citrus Psyllid populations. • Citrus Greening Survey o Pre Survey Results % Response (n=23) Not Little Very Questions Familiar Familiar Familiar Familiar Citrus greening disease 35 26 26 13 Citrus greening symptoms 22 52 13 13 Different strains of greening 61 26 9 4 79 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry disease Diagnostics methods to confirm greening 43 39 13 5 Insect vector that transmits greening disease 48 30 9 13 Life stages of Asian citrus psyllid 39 48 9 4 Pest reporting procedure 43 48 0 9 Fungal Bacterial Virus No Answer Is citrus greening a fungal, bacterial, or viral disease 22 9 48 21 The pre-survey (n= 23) showed that majority of the growers were not very familiar with the disease and its insect vector. When asked if citrus greening is bacterial, fungal or viral disease, only nine percent of the growers had a correct response that it is a bacterial disease. This survey showed that the citrus growers lack basic information about the citrus greening disease. We need to provide the growers with the information they required to manage this disease effectively. The post survey was sent out to 35 citrus growers in Louisiana and 29 growers responded to the survey. o Post survey results % Response (n=29) Not Little Very Questions Familiar Familiar Familiar Familiar Citrus greening disease 0 31 52 17 Citrus greening symptoms 3 24 48 24 Different strains of greening disease 14 24 55 7 80 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Diagnostics methods to confirm greening 10 28 48 14 Insect vector that transmits greening disease 3 28 59 10 Life stages of Asian citrus psyllid 3 31 45 21 Pest reporting procedure 7 24 48 21 Fungal Bacterial Virus No Answer Is citrus greening a fungal, bacterial, or viral disease 7 86 7 0 The post survey clearly showed a 77% increase in the grower’s knowledge of citrus greening being a bacterial disease. A 26% knowledge increase was observed regarding the citrus greening disease. Thirty five percent growers became more familiar with the symptoms of greening disease. Grower’s knowledge about different strains and diagnostic methods to confirm greening increased by 46% and 35%, respectively. The significant improvement regarding the familiarity of growers to the Asian citrus psyllid as a vector of greening (increased by 50%) and the life stages of Asian citrus psyllid (increased by 36%) was also observed during the post-survey as compared to pre-survey. During the pre-survey, it was observed that the growers (91%) were not aware of the greening reporting procedures and the post survey showed that 48% of growers became familiar and 12% became very familiar with the greening reporting procedure. The data from pre and post survey showed that our goal to increase the grower’s knowledge by 10% about the greening disease was fully accomplished. • Citrus grower meeting Meeting with citrus growers was conducted on March 11, 2014 in Belle Chasse, LA to discuss prevention and management of citrus greening. The meeting was attended by 34 citrus producers and methods such as budwood disease certification programs, regular 81 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry scouting and inspection, removal of HLB infected trees, insect proof screen houses and Asian citrus psyllid management were discussed with the audience. Beneficiaries • Provide a description of the groups and other operations that benefited from the completion of this project’s accomplishments. With the completion of this project, we expect the commercial citrus growers and commercial citrus nurseries to be greatly benefited. Louisiana does not have a citrus grower association. According to the 2013 Louisiana Ag Summary, there are 419 citrus producers in the state and citrus is grown on 744 acres. In Plaquemines where majority of citrus is grown, accounts for 535 acres. Here in this parish, there are 45 growers that have 40 or more trees for commercial production. Four commercial nurseries supply citrus trees with in Louisiana. In addition, they also supply citrus trees to neighboring states. In 2008, after citrus greening was first detected in Louisiana, several grower meetings were held to discuss the impact and management of this disease. The LSU AgCenter in collaboration with Louisiana Department of Agriculture and United States Department of Agriculture, started several campaigns to educate the commercial and homeowner citrus producers about citrus greening. During this project we did a pre and post survey to test the knowledge of the growers about this disease. We found that only 9% (n=23) were familiar with the fact that citrus greening is a bacterial disease. Forty eight percent and 22% thought that it is virus or fungal disease, respectively. Only 9% were familiar that citrus greening is transmitted by an Asian Citrus Psyllid (ACP) and 48% were not familiar with it. These numbers clearly indicate that growers had very little knowledge of this disease and its vector. A citrus greening and its vector training was organized in Belle Chasse on March 14, 2013 which was attended by 26 growers. During this training all the aspects about greening and ACP were discussed with the growers. Then during fall of 2013, a post survey was sent out to 35 growers and was asked same questions included in the pre- survey. A total of 29 growers responded to this post-survey. The post survey data showed that 86% (n=29) knew that citrus greening is a bacterial disease and 59% growers were now familiar that it is vectored by ACP. This shows that we were able to increase the grower’s knowledge about the disease and its vector and this knowledge will help the growers improve their management practices to manage this disease. • Clearly state the quantitative data that concerns the beneficiaries affected by the project’s accomplishments and/or the potential economic impact of the project. 82 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry According to 2013 Louisianan Ag Summary, the gross farm value of citrus is $5.4 million. After greening and two hurricanes (Gustav and Isaac), the citrus value has been significantly reduced from $9 million in 2009 to $5.4 million in 2013. During our project we found that the ACP and citrus tissue that we collected from different locations in Louisiana were all negative for citrus greening bacterium. This information can be used to determine if the 2008 detection was the only single episode of the disease occurrence in Louisiana. If this information is correct then the growers can be motivated to expand their commercial citrus production with best management practices. The total value of citrus nursery stock is estimated approximately $5 million and it is very important for these nurseries to continue their operation with up to date information on the whereabouts of this disease. Lessons Learned • Offer insights into the lessons learned by the project staff as a result of completing this project. This section is meant to illustrate the positive and negative results and conclusions for the project. Adverse environmental conditions, coordination with growers and parish government and occurrence of other important plants diseases significantly impacted the positive results and conclusions of this project. Although, we were able to collect Asian Citrus Psyllid (ACP) from several different location in Louisiana, but we observed that all these factors played an important role in the distribution of ACP population in Louisiana. In Louisiana on an average 3-4 flush (new growths) seasons occur and the peak activity of ACP occurs during late summer and mid fall. Unfortunately, Citrus Canker was positively detected in state on June 21, 2013 and as a result, we were unable to collect any psyllids due to movement restrictions and quarantine regulations. Citrus canker is a highly contagious disease and we did not want to spread the disease to non-infested sites. In 2013, we had a hard freeze which killed most of the new growth on citrus and it impacted the ACP population as well. Parish authorities in Plaquemines spray insecticides for ACP management in late spring and early fall and these dates were not confirmed. This resulted in reduction of ACP number that we expected to collect. In addition, we expected that the ACP will be widely distributed in Louisiana after it was first detected in 2008. On basis of this assumption, we selected two sites at Hammond and Baton Rouge to collect ACP. During the entire length of the project we did not find any psyllids at these two locations. Absence of ACP in these two locations is good, but for our purpose it was a bad decision to include these two locations. In 2012, we reported ACP from St. Bernard Parish, but during 2013 collection period no ACP were detected from this site. Unfortunately, we cannot control weather and occurrence of new diseases, but we learnt that more preliminary data was required to decide the ACP collection sites and coordination with Plaquemines Parish authorities to find out the exact dates of their spray program was necessary. 83 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry • Provide unexpected outcomes or results that were an effect of implementing this project. During this project ACP populations from Louisiana, Texas, California and Mexico were compared with 18S consensus sequences and we found that all four populations shared a common genetic background. We were expecting to see a difference between population from Texas and Louisiana compared to California and Mexico due to geographical locations. • If goals or outcome measures were not achieved, identify and share the lessons learned to help others expedite problem-solving. Goals and outcome measures were successfully achieved for this project. Contact Person Dr. Raghuwinder Singh (225) 578-4562 [email protected] Additional Information • Citrus Canker was detected in Louisiana in June 2013 and due to quarantine restrictions on the movement of any citrus related material, no new samples were collected. • Citrus greening positive trees have been found in Gentilly, LA on 3/5/2014, but no ACP was found on these positive trees. PROJECT SIX TITLE: Improving Sugarcane Beetle Management in Louisiana Sweet Potato Production Project Summary The sugarcane beetle, Euetheola humilis (Burmeister), is a polyphagous insect known to damage many crops in Louisiana, including sweet potato, corn, sugarcane, rice, pastures and strawberries. The insect was previously documented as a sporadic pest of sweet potato but has become more prevalent and problematic in recent years (Smith, 2006). A 84 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry 2011 survey of Louisiana sweet potato producers cited the sugarcane beetle as their number one pest management concern. Despite the documented pest status of the sugarcane beetle in many cropping systems, aspects of the biology and ecology of this insect remain poorly understood (Smith, 2006). Sweet potato producers have historically relied on prophylactic use of insecticides applied preplant and layby to manage soil insects (Smith et al., 2008). Unlike most soil insects feeding on sweet potato, it is the adult stage of the sugarcane beetle that inflicts damage (Hammond, 2002). The insects feed on other crops including corn and sugarcane in the spring, mate and lay eggs in late spring and develop over the summer months. It is the new or recently emerged generation that enters sweet potato fields prior to harvest (August – October) and causes damage. Damage has been inconsistent between years and between fields on affected farms. The proposed project aimed to increase the competiveness of sweet potato producers in Louisiana through enhanced insect pest management which will ultimately contribute to the long-term economic sustainability of the Louisiana sweet potato industry. Sweet potatoes are an important commodity in Louisiana, contributing over $117 million to the state’s economy in 2014. Approximately 13,000 acres of sweet potatoes were produced in Louisiana in 2011, the year prior to the project being funded. Soil insects feeding directly on the marketable roots have historically been one of the largest constraints facing Louisiana producers. These insects compromise the aesthetic appeal and marketability of the crop, thereby drastically reducing profitability for producers. The most prevalent soil insect threat during the five years preceding this project, was the sugarcane beetle. In 2010, over $1 million in losses was reported across the state due to damage caused by this insect. In severe cases, sugarcane beetle feeding renders roots unmarketable for both fresh market and processing sectors of the industry. It is paramount that research be conducted to improve management options for this insect in sweet potato. An integrated system-based research approach evaluating the biology of the sugarcane beetle, the diverse cropping systems in which sweet potatoes are produced and current insecticide technology will be beneficial as we move forward to develop an integrated pest management program for this insect in sweet potato and improve the economic sustainability of our industry. The landscape of Louisiana agriculture had changed dramatically during the last five years leading up to this project, especially in northern production regions where 75% of the state’s sweet potatoes are produced. Cotton acreage has dwindled while acreages of corn, soybeans and wheat have increased. With corn also being a preferred host of the sugarcane beetle (Smith, 2006), it is possible that the changing agricultural landscape contributed to escalated incidence of damage from this insect. In addition, many agronomic crops are now produced under minimum tillage practices. Crop residue left in fields from rotational crops may also be contributing to the escalating problem in sweet potato, as the beetle is documented to overwinter in pastures and unmanaged or fallow fields (Smith, 2006). 85 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry The insecticide use patterns of Louisiana sweet potato producers also changed about the same time, approximately 6 years ago. Producers had historically used Mocap® (ethoprop) and Lorsban® (chlorpyrifos) to manage soil insects; however, with the labeling and documented efficacy of Capture® (bifenthrin), many producers began using this product at a reduced price to manage numerous insect species. The sweet potato industry continues to be in a period of rapid change. Growth in the processing sector, emerging insect and disease problems and new varieties are changing the dynamics of the crop and are necessitating the need for research and outreach to help ensure sustainability. Several factors including growth in the processing sector and increased per capita consumption are fueling interest and growth in the industry but rising input costs and quality concerns have producers struggling to maintain profitability. With input cost exceeding $3,500 per acre in Louisiana it is critical that every sweet potato produced be of marketable quality. Improving pest management programs for sweet potato will improve the efficiency of sweet potato operations and increase the marketing potential of the crop. Additional information is needed for producers to implement a systems based integrated pest management program for the sugarcane beetle. Doing so will result in decreased costs and improved marketability of the crop, ultimately contributing to the sustainability of a $140 million specialty crop industry in Louisiana. Project Approach Objectives of the project were as follows: (1) Monitor sugarcane beetle activity patterns at several locations throughout Louisiana (2) Evaluate the field efficacy of labeled insecticides in leading commercial cultivar production systems to minimize damage caused by the sugarcane beetle and maximize sweet potato quality (3) Conduct laboratory bioassays to better understand the mechanism of documented efficacy of certain insecticides labeled for control of the sugarcane beetle (4) Evaluate the effects of surrounding cropping systems on damage from the sugarcane beetle (5) Rapidly disseminate research findings to the Louisiana Sweet Potato Industry (6) Evaluate impact of project activities on insect pest management programs for sweet potato The two principal investigators devoted a portion of their time to work on the goals and outcome measures of the project. In addition, a research associate and several transient/student workers assisted with project activities and reported directly to project directors. 86 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Sweet potato producers provided land and resources for on-farm activities and demonstration plots. Goals and Outcomes Achieved (1) Monitor sugarcane beetle activity patterns at several locations throughout Louisiana – this was done in all 3 years of the project – data provided. (2) Evaluate the field efficacy of labeled insecticides in leading commercial cultivar production systems to minimize damage caused by the sugarcane beetle and maximize sweet potato quality – this was done in all three years of the project – data provided (3) Conduct laboratory bioassays to better understand the mechanism of documented efficacy of certain insecticides labeled for control of the sugarcane beetle – this was done successfully in only one year of the project. Experiments were initiated in years 2 and 3 of the project but we could not obtain beetles for successful completion of these experiments. (4) Evaluate the effects of surrounding cropping systems on damage from the sugarcane beetle – this was done in year 2 and 3 of the project. A summary is provided. (5) Rapidly disseminate research findings to the Louisiana Sweet Potato Industry – all data collected through the 2014 crop year has been shared with the industry. Research completed in 2015 will be further summarized and presented to the industry during winter advisory and production meetings in 2015 and 2016. (6) Evaluate impact of project activities on insect pest management programs for sweet potato – a survey is being designed and will be disseminated to producers during our annual state meeting in January 2016 to evaluate current insect pest management programs in sweet potato, in light of information obtained through this project. Results have been and will continue to be disseminated to Louisiana producers and consultants and the Louisiana Sweet Potato Commission and Association meetings, by oral presentations at local and state industry meetings, visits to individual farming operations, trade industry newsletters, and LSU AgCenter electronic information dissemination networks (i.e., recommendation guides, newsletters). A distinct and measurable outcome of this project will be adoption of best management practices for management of the sugarcane beetle in sweet potato and maximization of quality and profit in the sweet potato industry in Louisiana. A set of recommendations developed during the life of the project will promote the adoption of best management practices. The best management practices for the sugarcane beetle will be incorporated into the extension publication describing the sugarcane beetle’s pest status in sweet potato. Best management practices related to insecticide efficacy, seasonal monitoring and cultural practices will be highlighted in this publication. A series of trade publications/extension bulletins will also be developed and disseminated to public and private sector stakeholders in the Louisiana sweet potato industry. This is a long term metric that is currently underway. An extension publication will be published 87 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry documenting the sugarcane beetle’s pest status in sweet potato and corn in Louisiana. Research findings will also be published in technical and scientific journals. Annual insecticide evaluations conducted in conjunction with this research have been published in Arthropod Management Test. In addition, a Louisiana Agriculture article was published highlighting the work associated with the third objective. See appendixes. Goals are in line with original objectives. Progress was limited with objective three due to reliance on natural insect populations for field infestation studies. Outreach objectives will be fulfilled completely with development and completion of extension publications highlighting best management practices for the sugarcane beetle and presentations delivered during December 2015 and January 2016. Data summary included below. 2013 Data Mechanism of documented efficacy of certain insecticides (Objective 3) A field experiment assessed the impact of Belay (preplant or layby timing) and Lorsban (preplant timing) on sugarcane beetle survival and injury to sweet potatoes. Sugarcane beetle mortality increased in Lorsban-treated plots whereas Belay applications were not associated with increased mortality. However, insecticides treatments did not decrease sugarcane beetle injury. This experiment suggests that the documented activity of Belay is caused by an antixenotic effect rather than increased mortality. Table X: Sugarcane beetle recovery success, sugarcane beetle mortality, and sugarcane beetle-associated root injury in an experiment using controlled infestations on sweet potato roots, Chase, LA, 2013. Treatment Probability of Probability of Probability of an recovering a recovering a dead injured root sugarcane beetle sugarcane beetle Untreated 0.81 0.03 0.17 Belay (preplant) 0.84 0.02 0.12 Belay (layby) 0.90 0.02 0.11 Lorsban 0.79 0.34 0.06 (preplant) F value 0.6 4.6 1.7 P < F 0.652 0.018 0.235 Table 1: Injury recorded in insecticide evaluations for sweet potato insect control, St Landry Parish, LA, 2013. Experiment 1 Experiment 2 88 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Treatment Timing Rate % Roots % Roots % Roots % Roots with (fl with insect with SCB with insect SCB gougesa oz/A) injurya gougesa injurya Untreated check -- -- 27.0ab 10.0ab 51.0a 9.0 Prepla Admire Pro 10 34.5ab 1.0bc 21.0bc 2.0 nt Admire Pro Layby 3.1 30.4ab 2.0bc 44.0ab 9.0 Prepla Belay 12 21.4ab 1.0bc 26.0abc 5.0 nt Belay Layby 12 20.0ab 0.0c 28.0abc 3.0 Prepla Brigade 19.2 33.3ab 4.1abc 29.0abc 4.0 nt Brigade Layby 19.2 31.0ab 13.0a 34.0abc 7.0 Prepla Brigadier 7.7 39.0a 2.0bc -- -- nt Brigadier Layby 7.7 35.5ab 5.0abc -- -- Prepla Lorsban 64 23.0ab 2.0bc 18.0bc 4.0 nt Lorsban+Admire Prepla 64+10 Pro nt 10.2b 1.0bc 8.0c 4.0 12 Belay Layby F value 2.4 5.1 4.7 1.2 P < F 0.033 <0.001 0.002 0.331 aMeans followed by the same letter are not different (Tukey’s HSD, α = 0.05) Insecticide Evaluation at Chase, LA location 2013: Overall insect pressure was low in this trial; damage in the non-treated plots was 7.5 and 0.6 percent for CB and SCB injury, respectively (Table 1. The proportion of cucumber beetle injured roots in treated plots did not differ from that in untreated check plots, however only the Admire pre-plant / Belay layby treatment resulted in significantly less cucumber beetle damage than the non-treated control. (Table 1). No significant differences in sugarcane beetle damage were detected between treatments. 89 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Table 1. Treatment/ Rate Formulation fl oz/acre Timing % SCB % CB Damage Damaged Roots Roots Admire Pro 10.5 preplant 0 0 c Admire Pro 3.5 layby 0.6 1.3 bc Belay 2.13 SC 12.0 preplant (infurrow) 0.6 3.8 abc Belay 2.13 SC 12.0 preplant (broadcast) 1.9 2.5 bc Belay 2.13 SC 12.0 layby 0.8 2.5 bc Lorsban 4 E 64.0 preplant 0.6 2.5 bc Lorsban 4 E 64.0 preplant 1.9 1.3 bc Belay 2.13 SC 12.0 layby Lorsban 4 E 64.0 preplant 0.6 3.8 abc Belay 2.13 SC 12 preplant Admire Pro 3.5 layby Lorsban 4 E 64.0 preplant 1.9 1.3 bc Admire Pro 10.0 preplant Belay 2.13 SC 12.0 layby Brigade 19.2 preplant 4.7 10.0 a Brigade 19.2 layby 0.6 10.0 a Admire Pro 10.0 preplant 1.4 0.0 c Belay 2.13 SC 12.0 layby Non-treated -- 0.6 7.5 ab 90 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Column means followed by the same letter are not significantly different, P=0.0320 (CB) (LSD, P > 0.05). No significant differences in SCB damage were detected between treatments. On-farm variety evaluation: Data were collected from an on-farm experiment conducted in Franklin Parish, LA to evaluate the susceptibility of selected sweet potato varieties to soil insect pests, including the SCB. A total of 11-21 roots were randomly selected from of each plot during harvest on 11/25/13. Susceptibility to insects was evaluated by recording the presence of insect injury on each root. Only numerical trends (P>0.05) for reduced injury in Murasaki and Beauregard were detected (Figure 1). Figure 1: SCB injury recorded in a sweet potato variety evaluation, Franklin Parish, LA, 2013. Sugarcane beetle flight activity monitoring: SCB activity was monitored continuously from early April to mid-November, 2013 at the LSU AgCenter Dean Lee Research Station in Alexandria, LA. Trapping revealed two peaks in flight activity (Figure 2). The majority of the spring generation flew between mid-April and mid-June whereas the majority of the late summer-fall generation flew between mid-September and late October. 91 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Sugarcane Beetle Pipe Study – summary of data for beetle recovery, mortality and injured roots. Generalized Linear Mixed Models with Binomial Distribution Proba finding SCB Binomial Type III Tests of Fixed Effects Effect Num Den DF F Pr > F DF Value Treatment 3 8.297 0.57 0.6516 Effect=Treatment Method=Tukey-Kramer(P<.05) Set=1 Treatment Mean Standard Letter Error of Mean Group 2 0.8972 0.04894 A 1 0.8368 0.06259 A 4 0.8149 0.06659 A 3 0.7934 0.07018 A Proba dead SCB Binomial Type III Tests of Fixed Effects Effect Num Den DF F Pr > F DF Value Treatment 3 15.45 4.57 0.0176 92 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Effect=Treatment Method=Tukey-Kramer(P<.05) Set=1 Treatment Mean Standard Letter Error of Mean Group 3 0.340 0.1383 A 4 0.025 0.02758 A 1 0.023 0.02613 A 2 0.021 0.02394 A Proba injured root Binomial Type III Tests of Fixed Effects Effect Num Den DF F Pr > F DF Value Treatment 3 8.974 1.7 0.2353 Effect=Treatment Method=Tukey-Kramer(P<.05) Set=1 Treatment Mean Standard Letter Error of Mean Group 4 0.166 0.03843 A 1 0.116 0.03243 A 2 0.109 0.03205 A 3 0.056 0.02467 A 2014 Data Sugarcane beetle flight activity monitoring (Objective 1) Sugarcane beetle flight activity was monitored with black light traps at the LSU AgCenter Dean Lee Research Station and Macon Ridge Research Station from spring to fall. Additional trapping was conducted on a commercial farm on the Evangeline/St Landry parish line, but onky during spring and early summer. Trapping revealed two periods of high flight activity, consistent with the literature. A first peak in flight activity was observed between late April and mid-May at the three locations. A second peak was observed in mid-September at the Dean Lee Research Station. However, flight activity during the fall of 2014 was relatively low compared to flight activity during the fall of 2013. 93 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Field insecticide efficacy evaluations (Objective 2) Two field experiments were conducted on a commercial farm in St Landry Parish to evaluate insecticides for management of sweetpotato soil insect pests, including the the sugarcane beetle. In addition to an untreated check, the same 8 insecticide treatments were assessed in each experiment. These insecticide treatments were the same as in “Experiment 2” conducted in 2013. In the first experiment, which was planted early, untreated plots exhibited 22% of roots with insect injury. Injury levels in untreated plots were not different from those observed in insecticide-treated plots, which sustained 13 to 41% insect injury. The majority of the observed injury was caused by larvae of cucumber beetles and whitefringed beetles. In the second experiment, which was planted late, the level of roots with insect injury was ≤ 5% regardless of insecticide treatment. Mechanism of documented efficacy of certain insecticides (Objective 3) The second year of the study assessing the impact of Belay and Lorsban on sugarcane beetle survival and injury to sweet potatoes was initiated at the Sweet Potato Research Station and Dean Lee Research & Extension Center. The experimental methodology involved the release into PVC enclosures of sugarcane beetles caught at black lights. However, the experiments could not be completed because sugarcane beetles needed for artificial infestation of PVC enclosures could not be collected. Evaluation of the effects of surrounding crops on damage from the sugarcane beetle (Objective 4) Twelve flight intercept traps were developed, built, and deployed at the LSU AgCenter Sweet Potato Research Station during the summer of 2014 to conduct a pilot study. Three pairs of traps were placed in a sweet potato field. Each pair of trap faced either a soybean field, a fallow wheat field, or a bermudagrass pasture. Traps were monitored weekly from Early July to late October, 2014 to determine the impact of surrounding landscape on sugarcane beetles infesting sweet potato fields. However, sugarcane beetle flight activity was extremely low during the experiment and sugarcane beetles were not caught in the traps. 2015 Data Sugarcane beetle flight activity monitoring (Objective 1) Sugarcane beetle flight activity was monitored with black light traps at the LSU AgCenter Dean Lee Research Station and Macon Ridge Research Station (Figure 1). Consistent with trapping data collected during the previous two years of the project, sugarcane beetle populations exhibited a peak in flight activity from early spring into early summer. In 2015, significant flight activity during late summer and early fall was not detected at either trapping location. 94 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry (A) (B) Figure 1: Sugarcane beetle flight activity recorded at the (A) Dean Lee Research Station and (B) Macon Ridge Research Station, 2015. Field insecticide efficacy evaluations (Objective 2) Two experiments were conducted in 2015 on a farm in St Landry Parish, LA to evaluate insecticides for management of sweet potato soil insect pests, including the sugarcane beetle. In addition to an untreated check, 5 and 8 insecticide treatments were evaluated in Experiment 1 and 2, respectively (Table 1). A total of 15-25 roots were randomly selected from the 2 center rows of each plot during harvest on 10/22/15 (Experiment 1) and 11/24/15 (Experiment 2). Insecticide efficacy was evaluated by recording the presence of insect injury on each root for Experiment 1. Samples from Experiment 2 have not been processed at the time of this report (Table 1). Insect-caused holes and gouges were observed on 37% of the roots collected in the untreated check plots of Experiment 1 (Table 1). The majority of the injury was holes caused by larvae of cucumber beetles, with gouges caused by sugarcane beetle, white grubs, and whitefringed beetles only being observed on 2 to 9% of the roots across 95 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry insecticide treatments. The low sugarcane beetle pest pressure was consistent with the reduced flight activity observed using light traps. The proportion of injured roots in treated plots did not differ from that in untreated check plots (Table 1). Table 1: Injury recorded in insecticide evaluations for sweet potato insect management, St Landry Parish, LA, 2015. Experiment 1 Experiment 2 Treatment Timin Rate % Roots % Roots % Roots g (fl with insect with with insect oz/A) injury gouges injury Untreated check -- -- 37.0 2.0 Prepla Admire Pro 10 32.0 3.0 nt Admire Pro Layby 3.1 -- -- Prepla Belay 12 35.0 9.0 nt Samples are Belay Layby 12 -- -- being Prepla processed at Brigade 19.2 27.0 2.0 nt the time of this report Brigade Layby 19.2 -- -- Prepla Lorsban 64 21.0 6.0 nt Lorsban+Admire Prepla 64+10 Pro nt -- -- 12 Belay Layby Lorsban+Admire Prepla 64+10 17.0 2.0 -- Pro nt F value 1.5 1.0 P < F 0.250 0.441 Two additional experiments were conducted in 2015 Franklin Parish, an early planting date trial and a late planting date trial, to evaluate labeled insecticides for control of the sugarcane beetle. Injury ratings for sugarcane beetle in the two trials were too low to report. Total yield estimates and cucumber beetle damage are reported (Table 2 and 3). Table 2: Yield and insect injury recorded in an early planted insecticide evaluation for sweet potato insect management, Franklin Parish, LA, 2015. Treatment/ Rate Formulation fl oz/acre Timing Total % CB 96 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Yield Damaged (Bu/A) Roots Admire Pro 10.5 preplant 1151 1.0 Admire Pro 3.5 layby 964 6.0 Belay 2.13 SC 12.0 preplant (infurrow) 931 8.0 Belay 2.13 SC 12.0 layby 989 7.0 Lorsban 4 E 64.0 preplant 1025 16.0 Lorsban 4 E 64.0 preplant 1041 7.0 Admire Pro 10.5 preplant Belay 2.13 SC 12.0 layby Brigade 19.2 preplant 1182 2.0 Brigade 19.2 layby 1097 18.0 Non-treated -- 1012 7.0 No significant differences were detected between treatments for yield or insect damage. P=1.562 (CB) (LSD, P > 0.05). Table 3: Yield and insect injury recorded in a late planted insecticide evaluation for sweet potato insect management, Franklin Parish, LA, 2015. Treatment/ Rate Formulation fl oz/acre Timing Total % CB Yield Damaged (Bu/A) Roots Admire Pro 10.5 preplant 1039 3 Admire Pro 3.5 layby 979 11 Belay 2.13 SC 12.0 preplant (infurrow) 1062 6 Belay 2.13 SC 12.0 layby 1038 3 Lorsban 4 E 64.0 preplant 1134 10 97 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Lorsban 4 E 64.0 preplant 1054 5 Admire Pro 10.5 preplant Belay 2.13 SC 12.0 layby Brigade 19.2 preplant 972 5 Brigade 19.2 layby 935 11 Non-treated -- 979 16 No significant differences were detected between treatments for yield or insect damage. P=1.446 (CB) (LSD, P > 0.05). Variety evaluation (Additional Objective) A field experiment evaluating the susceptibility to soil insects in 8 sweet potato genotypes was conducted at the Dean Lee Research Station in 2015. Experimental plots were planted on 6/22/15 and no insecticide treatments were applied until harvest on 10/21/15. For each plot, 15-25 roots were randomly selected and rated for insect injury. Sugarcane beetle injury was not observed. However, holes caused by larvae of cucumber beetles were abundant. The resistant standard ‘Murasaki’ sustained levels of injury 35% lower than the susceptible standard ‘Beauregard’ (Table 4). The commercial ‘Bellevue’ and experimental ’13-84’ were not different from Murasaki and Beauregard. The commercial ‘Orleans’ and experimental ’13-111 A’, ’13-93’, and ’11-35’ were as susceptible to insect injury as Beauregard. Table 4: Cucumber beetle injury recorded in a sweet potato variety evaluation, Dean Lee Research Station, LA, 2015. Genotype % Roots with cucumber beetle injurya Beauregard 99.0a Murasaki 64.0b Bellevue 83.6ab Orleans 96.0a 13-111 A 99.0a 13-84 95.0ab 13-93 99.0a 11-35 99.0a F value 3.4 P < F 0.012 aMeans followed by the same letter are not different (Tukey’s HSD, α = 0.05) Mechanism of documented efficacy of certain insecticides (Objective 3) A field experiment aiming to examine the effect of Belay (preplant or layby timing) and Lorsban (preplant timing) on sugarcane beetle survival and injury to sweet potatoes under 98 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry controlled infestations was initiated at the Dean Lee Research Station on 6/22/15. The experimental methodology involved the release into PVC enclosures of sugarcane beetles caught at black lights. However, sugarcane beetle trap catches were extremely low and sugarcane beetle releases could not be conducted. Sampling of the experimental area between 7/31/15 and 10/6/15 revealed that banded cucumber beetles occurred at densities substantially greater than the currently recommended threshold of 2 cucumber beetles per 100 sweeps (27 to 200 per 100 sweeps). Because foliar insecticides were not applied to manage cucumber beetles, 25 roots were harvested for each plot on 10/21/15 and rated for cucumber beetle injury using a severity index (0 = no injury, 1 = 1-5 holes or scars, 2 = 6-10 holes or scars, 4 = > 10 holes or scars). A single application of Belay, preplant or at layby, assisted in decreasing the end-of-season percentage of roots with cucumber beetle injury by over 50% (Table 5). In addition, even when roots were injured by cucumber beetles, there was some evidence that the Belay assisted in decreasing the severity of injury (Table 5). The preplant Lorsban application did not provide control of cucumber beetles. Table 5: Cucumber beetle injury recorded in a sugarcane beetle insecticide study, Dean lee Research Station, LA, 2015. Treatment % Roots with cucumber beetle Severity index for roots injurya with cucumber beetle injury Untreated 39.0b 1.9 Belay (preplant) 41.0b 1.8 Belay (layby) 89.0a 3.0 Lorsban (preplant) 84.8a 2.7 F value 17.2 3.4 P < F < 0.001 0.068 aMeans followed by the same letter are not different (Tukey’s HSD, α = 0.05) Evaluation of the effects of surrounding crops on damage from the sugarcane beetle (Objective 4) Twenty-one flight intercept traps were deployed in three sweet potato fields in Franklin Parish during expected sugarcane beetle peak flight activity in late summer-early fall. The goal of this study was to determine how adjacent habitats could impact sugarcane beetle infestations in a particular sweet potato field. In each sweet potato field, 2 or 3 edges facing an adjacent grassy area, corn field, or sweet potato field were selected so that each adjacent habitat was replicated twice for the study. For each edge, 3 flight intercept traps were placed 50-100 ft apart and monitored weekly for 3 weeks. Consistent with the reduced flight activity observed using black light traps, no sugarcane beetles were collected. Beneficiaries There are about 65 commercial sweet potato producers in Louisiana. Research efforts will help provide needed information on management of the sugarcane beetle to the entirety of the Louisiana sweet potato industry, including producers that are growing for 99 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry the fresh market and processing sectors. This information will also be useful to numerous consultants throughout. The strategies developed as a result of this research may lead to a more efficient and effective integrated pest management programs for the sugarcane beetle, which will have a positive direct impact for sweet potato producers in Louisiana and throughout production regions of the United States where the sugarcane beetle has been documented as a significant pest. Additionally, citizens of Louisiana and beyond are anticipated to benefit from this research as it may contribute to increased profitability of sweet potato producers thereby directly and indirectly contributing to economic growth. The project aimed to increase the competiveness of sweet potato producers in Louisiana through enhanced insect pest management which will ultimately contribute to the long- term economic sustainability of the Louisiana sweet potato industry. Sweet potatoes are an important commodity in Louisiana, contributing over $117 million to the state’s economy in 2014. Through this research project we have reaffirmed our current insecticide recommendations for the sugarcane beetle and learned more about emergence patterns and damage potential for this insect in sweet potato in Louisiana. Lessons Learned This project has provided the resources necessary to conduct research on a high priority knowledge gap as identified by the Louisiana Sweet Potato Industry. As stated previously, principal investigators were able to further understand the biology of the sugarcane beetle and have further documented the damage potential and the sporadic occurrence of this insect in sweet potato. We have also reaffirmed our current insecticide recommendations for this insect. Researchers gained insight into the efficacy of neonicotinoid insecticides for management of cucumber beetles in sweet potato. We learned that producers are benefiting from season long reductions in damage from applications of Belay and Admire. In treatments devoid of any foliar insecticide applications, researchers realized over a 40% decrease in damage. Often with in-field entomology research the project becomes limited based on natural insect populations. Though the project accomplished a great deal and elucidated information for management of the sugarcane beetle, much more could have been discerned and discovered if sugarcane beetle populations would have been greater during the course of the project. Contact Person Tara Smith 318-435-2155 318-557-9501 [email protected] 100 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Additional Information Provide additional information available (i.e. publications, websites, photographs) that is not applicable to any of the prior sections. Managing Sugarcane Beetles in Louisiana Sweet Potatoes Figure 1. Adult sugarcane beetle. Photo by Jerry Lenhard Figure 2. Sweet potatoes with sugarcane beetle injury. Photo by Tara P. Smith 101 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Figure 3. PVC pipe used to contain beetles. Photo by Tara P. Smith Figure 4. Test area with PVC pipes. Photo by Tara P. Smith Figure 5. Plastic tub with sweet potato plant and surrounding soil. Photo by Tara P. Smith 102 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Tara P. Smith, Julien M. Beuzelin, Theresa F. Arnold and Dana M. May Sweet potatoes are an important agricultural commodity in Louisiana, with a total production value exceeding $85 million in 2013. Soil insect pests, which reduce root yield and quality, are often cited as one of the most limiting factors affecting production. The sugarcane beetle (Figure 1) has emerged as the primary Figure 6. Device used to sift target of soil insect management programs for sweet soil to recover beetles. Photo by Tara P. Smith potato producers in Louisiana during the past 10 years. Unlike most soil insects feeding on sweet potatoes, only adult sugarcane beetles cause crop damage. These adult beetles feed on sweet potatoes late during the growing season by creating gouges on the surface of the roots (Figure 2). Because sweet potatoes are an aesthetic crop, minimal damage negatively affects marketability of the roots. Figure 7. Sweet potato root Limited options are available to manage sugarcane harvested from PVC enclosure beetles in sweet potatoes. The insecticides Belay with sugarcane beetle feeding injury. Photo by Tara P. Smith 2.13SC (clothianidin) and Brigade 2EC (bifenthrin) are presently labeled for soil applications preplant or approximately three weeks after planting. Belay can control the sugarcane beetle and is currently Figures 8-10 recommended. Root injury has been reduced in field plots treated with these and other labeled insecticides; however, results have varied 103 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry among locations and production years. Thus, there is a need to better document activity of clothianidin and other insecticides of interest to refine sugarcane beetle management recommendations. A study conducted in 2013 at the LSU AgCenter Sweet Potato Research Station in Chase examined the efficacy of selected insecticide treatments against controlled sugarcane beetle infestations in sweet potatoes. All insecticides evaluated in the study are labeled for use on sweet potatoes in Louisiana and were applied to experimental plots prior to planting or 20 days after planting. Sweet potato transplants were planted at a rate of one plant per foot of row, consistent with commercial practices. Insecticide treatments were applied to experimental plots with a carbon-dioxide-charged spraying system calibrated to deliver 20 gallons per acre. Polyvinylchloride pipes 12 inches in diameter and 15 inches tall were placed 3 inches into the soil around individual sweet potato plants in each plot (Figures 3 and 4). Adult sugarcane beetles were collected using black lights during September and were maintained on sweet potato roots until needed for infestations. Four sugarcane beetle adults were individually placed on each plant surrounded by a pipe in each plot. Following this mechanical infestation, pipes were covered with a mesh screen cap secured by a rubber band. Feeding injury to the sweet potato roots, number of beetles recovered and beetle mortality were evaluated 10 days after insect release began by harvesting whole plant samples. Each sweet potato plant and surrounding soil contained within the pipe were placed into a plastic tub (Figure 5). All of the soil was sifted (Figure 6) to recover beetles. Each sweet potato root (Figure 7) was also examined for the presence of feeding injury. The majority of sugarcane beetles – 79-90 percent – were recovered (Figure 8). Injury ranged from 6 104 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry percent to 17 percent of sweet potato roots with sugarcane beetle feeding (Figure 9). However, differences in injury were not detected for any insecticide treatment evaluated in this study. Beetle mortality ranged from 2 percent to 34 percent, with the Lorsban 4E treatment resulting in significantly higher beetle mortality (Figure 10). Belay applications were not associated with increased mortality. Occurrence of and damage incurred from sugarcane beetles in sweet potatoes are highly erratic. The insecticides evaluated in the study have been shown to significantly reduce sugarcane beetle damage in naturally infested small-plot field studies. In field trials conducted from 2011 to 2014, sugarcane beetle damage ranged from zero to 40 percent in 2011, 12-57 percent in 2012, zero to 5 percent in 2013 and zero to 12 percent in 2014. The current study design allowed researchers to evaluate sugarcane beetle damage in sweet potatoes in a contained system. Insufficient numbers of sugarcane beetles were collected during 2014, which prevented successful replication of the study. LSU AgCenter scientists plan to use the current design to evaluate insecticide management options for the sugarcane beetle in sweet potatoes at the Sweet Potato Research Station and the Dean Lee Research and Extension Center in subsequent years. Studies evaluating cropping systems adjacent to sweet potatoes and alternative monitoring techniques for the sugarcane beetle are also in progress. Results from these studies will be integrated into a multidisciplinary pest management program for the sugarcane beetle in sweet potatoes. Tara P. Smith is associate professor and research coordinator at the Sweet Potato Research Station, Chase, La.; she is also the director for the Northeast Region. Julien M. Beuzelin is an assistant professor at the Dean Lee Research and Extension Center, 105 Specialty Crop Block Grant Program – Louisiana Department of Agriculture & Forestry Alexandria, La. Theresa F. Arnold is research associate and graduate student at the Sweet Potato Station, and Dana M. May is research associate at Dean Lee. (This article was published in the winter 2015 issue of Louisiana Agriculture.) 106