Final report for FNC22-1338

FNC22-1338 (project overview)

Project Type: Farmer/Rancher

Funds awarded in 2022: $19,437.00

Projected End Date: 06/15/2025

Grant Recipient: Solid Ground Farm

Region: North Central

State: Ohio

Project Coordinator:

Weston Lombard

Solid Ground Farm

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Project Information

Description of operation:

Solid Ground Farm is a 60 acre diversified small farm and education center with a focus on agroforestry and homescale livestock. We are particularly interested in improved native and naturalized fruit and nut crops to feed people and livestock and have several orchards and forest gardens of various sizes and ages producing everything from Chinese chestnuts to mulberries. The farm is 12 years old and also houses a summer camp, a nature-based elementary school, and a community garden.

Summary:

Our native red mulberry, Morus rubra—a fruit-producing tree with delicious, nutritious, complex-flavored fruit, high production potential, innate pest/disease resistance, and rapidly growing commercial and home production interest—is slowly fading from its native range due to the rapid spread of the invasive Morus alba. Morus alba was imported in the 1600s and planted by the millions across the eastern seaboard in a failed attempt to create a domestic silk industry. Trees quickly escaped from these orchards and have since naturalized across the country. These invasive trees release large amounts of wind-dispersed pollen that readily pollinate the native trees, producing hybrid offspring that are steadily and irrevocably replacing pure Morus rubra.

In order to address this slow-motion extinction by hybrid replacement, we propose to find, propagate, and establish preserves and breeding orchards of high-quality, fruit-producing Morus rubra specimens with commercial potential. By holding a national mulberry Search and Rescue Contest, we plan to crowdsource the best Morus rubra trees growing wild, in orchards, and nursery rows across the country. Entries will be genetically tested, rated for potential as commercial cultivars, and the winning trees named and made available to farmers, nurseries, researchers, and fruit enthusiasts throughout the North Central region.

Through this project we created a Morus rubra identification guide and helped the public to identify and share samples from mulberry trees suspected of being pure Morus rubra. We received over 100 fresh leaf samples from around the country. Samples were sent to the Ohio University Genomics Facility where they were frozen and then the DNA extracted. Samples were then used to begin constructing a Morus rubra genome by researchers at the Los Alamos National Laboratory (this process is ongoing due to the complexity of the genome). This information was then cross-referenced with the existing Morus alba genome and each individual sample was analyzed and results compiled. Because of the complexity of the project and the lack of an existing complete rubra genome, the results are not necessarily indicative of complete purity of species, but do show with a high degree of accuracy whether a sample is rubra, alba, or a hybrid, as well a good inidcation of the degree of possible hybridization. Results for instance indicate that Illinois Everbearing, a popular mulberry considered a hybrid, has mostly rubra characteristics but does contain a trace of alba. For the results of the study, we classified specimens by the closest obvious fit and marked those with mainly rubra characteristics as "Rubra" and those with obvious high alba influence as "Hybrids". A closer look at the numbers, however, can tell us the degree of hybridization of each specimen.

While imperfect, the results do indicate that many pure or nearly pure rubra trees do still exist in different regions across the country, and while further analysis will be needed to determine complete purity, we are moving in the right direction and have found specimens with good fruit production that can be considered native and are definitely worth growing as food producing trees.

Project Objectives:

Hold a contest to find productive Morus rubra specimens with commercial potential and perform genetic testing of advertised "Morus rubra" cultivars (often mislabeled Morus alba trees that have red fruit) to establish verified native sources.
Name and release several new Morus rubra cultivars suitable for orchard production.
Propagate and establish a preserve and breeding orchard in order to provide verified native mulberry plant material to nurseries, farmers, conservation workers, researchers, and landowners throughout the North Central region.
Promote the value, usefulness, and profitability of growing native mulberry trees through field days, website and social media promotions, and a scholarly journal.

Cooperators

Click linked name(s) to expand/collapse or show everyone's info

Nigel Daniels - Technical Advisor (Researcher)
nigel.daniels1@gmail.com
Genomics Consultant
Jonathan Gesualdi - Technical Advisor (Researcher)
jonathan@thenakedoaktreefarm.com
Morphological Assessment
The Naked Oak Tree Farm

Research

Materials and methods:

The goal of this project is to identify superior Morus rubra specimens suitable for fruit production. Then genetically test them to establish pure native sources of mulberry to share with the general public. To achieve this we have taken a crowdsourcing approach and created a Facebook event promoting a Mulberry Search and Rescue Contest. We are offering free genetic testing to participants and help developing connections with nurseries and other buyers of mulberry cuttings and scionwood for those interested. We will then buy propagation materials from the test entrants and clone the trees to start a research/demonstration/breeding orchard.

The contest has gone well and has been promoted heavily on various interest groups and online forums. We found some great potential trees, but have been delayed in the testing process. We based our initial plan on a Canadian study that established genetic markers for wild Morus rubra trees up north. However, upon deeper review of the study methods, our geneticist consultant suggested that the number of markers and the type of test used (RAPD) were insufficient for our goals of establishing definitive purity of our source material. As a result, we have spent considerable time researching alternative testing plans and have come up with a new plan utilizing GBS testing that will provide more accuracy. This project is particularly challenging because, unlike more common trees, the genome for Morus rubra has not yet been mapped to provide a basis for easy comparison, so we are essentially building the map as we go.

This revamping is resulting in a delay and relaunch of the contest.

After relaunch we now have 100 promising samples from around the country and have chosen a half dozen of them that appear most likely to be pure Morus rubra specimens for our first round of testing. These samples will be tested and reviewed to create a reference genome and establish markers for identifying future rubra specimens. After this is established, the remaining samples will be compared to this reference and cross-referenced against the Morus alba genome to establish if samples are hybrids or pure rubra specimens. This process is new and has taken time to establish, resulting in a further delay of our results. We have just been approved for an 11-month extension to finish the project.

The project has expanded to include contributions from the Ohio University Genomics Lab, Nigel Daniels, Los Alamos National Laboratory (LANL), and Jonathan Gesualdi of The Naked Oak Tree Farm, and additional information will be added to this report as it becomes available. We have now completed the first round of testing and ou can read about the methodology and technical results from Nigel Daniels here.

In addition to the genetics test performed, we have partnered with, Jonathan Gesualdi, who has developed a morphological assessment and key using microscpopic viewing of trichome density and petiole groove/vascular bundle orientation to determine mulberry species. Jonathan is currently cross checking our genetic work to validate his process and refine our results. Information from his finding will be added to this report as it becomes available as well as on his website at: https://thenakedoaktreefarm.com/red-mulberry

From Jonathan: The above photo is of the abaxial view of sample #58 leaf where we are establishing a new trichome density range to differentiate hybrids vs pure rubra. This is based on the original paper Asymmetrical introgression by K. S. BURGESS, et.al. They proved that trichome density is a statistically significant trait that can be used to differentiate hybrids vs pure rubra. However, their mythology was unclear to replicate their observations. After conferring with the authors we decided that a different method was necessary what would be easier to replicate by anyone with a microscope and will be fully detailed in a key with instructions we would like to distribute to those seeking to tell pure rubra from hybrids visually.

This image focuses on the petiole morphology. This is an existing trait already included in the "More on Muddled Mulberries" paper that partners are performing on the sample set to confirm its accuracy and perhaps expand on the morphological differences in the vascular bundles.

This image is one that Paul and Julian are using in their soon to be published follow-up paper with discussion specifically on the petiole/leaf junction morphology and other expanded morphological assessments.

Photo credit: Paul Adanick

Jonathan is still accepting samples to test and can be reached at: Jonathan@TheNakedOakTreeFarm.com

Creating a complete Morus rubra genome proved to be outside the scope of this project, however, the momentum has been created and we hope to have more information to share in the coming years.

Research results and discussion:

Genetic testing has revealed the likely existence of many pure or nearly pure Morus rubra trees across the United States, and while based on current information, we cannot yet say with 100% accuracy which samples are entirely native, we have a good sense and are happy to share the results below. As more information becomes available and the genome is fully assembled, testing will become more and more accurate.

The results of our research can be viewed below. We are still assessing who from this list is interested in sharing scionwood from their trees and will soon add a document with a list of potential sources of cuttings for those interested in purchasing Morus rubra grafting material.

Results (from report by Nigel Daniels)
Read full report with methodologies here.

Ct values for each sample were compared for differences between detection of the species-specific genomic regions labeled Rub232, Rub236, and Alb233. Differences of ~10 cycles between specific-region Ct values were used as an indication of whether a sample was likely M. rubra, M. alba, or a hybrid. A lower Ct value indicates a higher quantity of genomic DNA containing the region targeted by that primer pair present in the reaction. Thus, values of ~18-21 for the Rub regions and >~28 for the Alb region indicate a likely M.rubra species identification. Values opposite to this indicate likely M.alba, whereas ~18-21 values for any combination of Rub and Alb regions indicate a likely hybrid.

Table 2. Summary of Quantitative PCR Threshold Cycle (Ct) values for the species-specific genomic regions Rub232, Rub236, and Alb233 produced from Mulberry leaf genomic DNA samples. “Indicated Species” denotes the species of the sample based on the regions tested but does not absolutely guarantee the absence of DNA from other species in untested regions.


Sample ID	Submitted by:	Rubra232 Ct	Rubra236 Ct	Alba233 Ct	Indicated Species
1	Jamie Belcourt, Conway AR	19.02	18.99	28.37	Rubra
2	Mark Travis, Gainesville FL, Williston	24.48	24	34.35	Mostly Rubra
3	Mark Travis, Gainesville FL, Katy	20.56	20.99	36.5	Rubra
4	Mark Travis, Gainesville FL, Barr Hammock	19.28	19.17	29.85	Rubra
5	Kathy Jacobson, Stewart OH, Gate	21.98	21.95	21.78	Hybrid
6	Kathy Jacobson, Stewart OH Stem at base of big tree	20.61	20.15	20.15	Hybrid
7	Peaceful Heritage Nursery, Stanford KY, Queen Luscious	19.42	20.02	19.94	Hybrid
8	Peaceful Heritage Nursery, Stanford KY 40484, Varaha	20.45	19.91	27.44	Mostly Rubra
9	Dan Greenberg, Matthews NC,	19.84	19.32	28.12	Rubra
10	Dan Greenberg, Matthews NC	19.98	19.44	31.97	Rubra
11	Michael & Keary Mariannino, Charlotte Court House VA	24.26	24.27	37.52	Rubra
12		Duplicate sample
13	Jay McGrath, Dayton OH	20.17	20.51	25.84	Hybrid
14	L. Eubank, Elizabethtown IN	20.84	20.11	20.15	Hybrid
15	Elizabeth Reznar, Lebanon OH	21.85	21.61	32.48	Mostly Rubra
16	Lydia Nichols, Chesterfield VA	21.73	21.67	23.87	Hybrid
17	David Snyder, Johnson County, IN	19.1	19.03	30.02	Rubra
18	Hunter	None	35.73	19.94	Alba
19	Leslie Wade "Hem & Holler", Ellijay GA	18.83	18.71	30.47	Rubra
20	Leslie Wade "Ever Wilder Farm", Ellijay GA	18.92	18.81	30.08	Rubra
21	Darren, Locust Grove E	18.82	18.82	29.72	Rubra
22	Darren, Locust Grove W	19.1	18.94	29.68	Rubra
23	Darren, Black Bramble ♂&♀	19.52	19.06	31.31	Rubra
24	Darren, Open Field	18.41	17.77	28.36	Mostly Rubra
25	Mike Hater 1	None	None	None	Unknown
26	Mike Hater 2	18.91	18.42	29.25	Rubra
27	Katarina Midelfort, Purplewood 1	18.87	18.58	29.8	Rubra
28	Katarina Midelfort, Purplewood 2	Duplicate sample
29	Katarina Midelfort, Stockdell 1	18.73	18.3	29.38	Rubra
30	Katarina Midelfort, Stockdell 2	Duplicate sample
31	James Arnold, Leland NC, Sample 1	18.55	18.64	30.69	Rubra
32	James Arnold, Leland NC, Sample 2	19.08	19.04	31.11	Rubra
33	Caryl Orr, Cottendale AL, Red Mulberry,	18.37	18.48	30.26	Rubra
34	Weston Lombard, Woodward Rubra	20.11	20.02	19.89	Hybrid
35	Weston Lombard, Liars corner Rubra	18.22	18.2	29.55	Rubra
36	Weston Lombard, Tartarica Alba #1	18.3	33.33	17.03	Hybrid
37	Weston Lombard, Tartarica Alba #2	32.52	33.51	19.33	Alba
38	Weston Lombard, Alba #3 Seedling	32.99	33.89	20.93	Alba
39	Weston Lombard, Alba #4 Seedling	None	30.2	17.38	Alba
40	Weston Lombard, Silkhope Rubraxalba	18.99	19.1	18.76	Hybrid
41	Weston Lombard, Maulticulis	18.19	29.07	18.53	Hybrid
42	Weston Lombard, Shelli	33.89	31.21	18.86	Alba
43	Weston Lombard, nursery bought rubra seedling	19.91	32.96	19.28	Hybrid
44	Weston Lombard, Rubra	18.38	17.92	28.59	Mostly Rubra
45	Weston Lombard, IE	18.37	18.13	25.79	Mostly Rubra
46	Weston Lombard, Kip Parker	19.61	18.98	19.19	Hybrid
47	Weston Lombard, World's Best	18.68	32.71	17.9	Hybrid
48	Weston Lombard, Oscar Rubrax Alba	19.5	30.19	18.1	Hybrid
49	Weston Lombard, SGF Alba	33.09	31.83	18.98	Alba
50	Weston Lombard, Terrace Alba	None	33.66	22.28	Alba
51	Weston Lombard, Terrace Rubra	20.09	19.9	27.29	Mostly Rubra
52	Weston Lombard, nursery Rubra seedling	33.14	30.55	19.26	Alba
53	Weston Lombard, Rubra seedling	33.7	33.87	18.13	Alba
54	Weston Lombard, Ukranian giant Alba	33.17	32.25	18.36	Alba
55	Weston Lombard, nursery Seedling	18.5	28.89	18.26	Hybrid
56	Vicki Shaub, Nashville TN 37211	19.24	18.77	30.52	Rubra
57	Vicki Shaub, Nashville TN 37211	Duplicate sample
58	Vicki Shaub, Nashville TN 37211	Duplicate sample
59	Benny Becker, Beckley WV, "Gorgeous"	19.44	18.99	19.14	Hybrid
60	Varaha, Peaceful Heritage Nursery	19.6	19.18	27.08	Mostly Rubra
61	Spillway, Lucky Pittman	19.17	18.07	29.57	Rubra
62	Cindy Redding, Mulberry Apiary	36.05	32.82	19.08	Alba
63	P Pitre Byron, Gray LA, Mature tree	19.49	18.86	35.6	Rubra
64	P Pitre Byron, Gray LA, Mature tree	Duplicate sample
65	P Pitre Byron, Gray LA , Sapling #1	19.58	19.61	31.55	Rubra
66	P Pitre Byron, Gray LA, Sapling #2	33.6	21.84	20.41	Hybrid
67	Elena Obradovich, Slemp KY 30+ yrs old, fruit producer	18.8	18.53	29.92	Rubra
68	James Garlanger, Oviedo FL, Bruce's Red	19.68	19.42	30.49	Rubra
69	James Garlanger, Oviedo FL, Rootstock seedling	20.58	32.86	19.89	Hybrid
70	James Garlanger, Oviedo FL, Oviedo H	19.33	19.23	31.04	Rubra
71	James Garlanger, Oviedo FL, Oviedo #1	19.56	19.39	30.93	Rubra
72	James Garlanger, Oviedo FL, Oviedo #2	20.89	20.39	32	Rubra
73	James Garlanger, Oviedo FL, Farm #1	19.73	19.22	31.33	Rubra
74	James Garlanger, Oviedo FL, Farm #2	18.79	18.43	28.94	Rubra
75	James Garlanger, Oviedo FL, Farm #3	19.69	19.14	30.91	Rubra
76	James Garlanger, Oviedo FL, Farm #4	20.34	19.7	31.53	Rubra
77	Marco Contino, Raleigh NC, Adeline's Mulberry	19.65	19.51	31.66	Rubra
78	David Snyder, Ortet, Smith Valley Mulberry,	20.27	20.38	29.57	Rubra
79	Zaros #1	18.52	18.49	30.84	Rubra
80	Zaros #2	18.77	18.7	30.66	Rubra
81	Zaros #3	19.25	18.91	31.19	Rubra
82	Zaros #4	18.85	18.77	29.68	Rubra
83	Zaros #5	18.52	18.18	30.25	Rubra
84	Chen Chang, Lebanon G, Miss Kim	19.89	32.95	18.88	Hybrid
85	Chen Chang, Lebanon GA, Robert	20.72	31.06	19.51	Hybrid
86	Chen Chang,, Lebanon GA , Greenwich	19.97	19.45	22.6	Hybrid
87	Chen Chang, Lebanon GA, Lucile	19.18	18.54	21.96	Hybrid
88	LLP H. Hopkinsville KY, Harmony Grove (East), ♂ M.rubra?	20.7	20.22	20.52	Hybrid
89	LLP H Hopkinsville KY Island Cove, Old ♀ M.rubra	19.01	18.87	30.74	Rubra
90	LLP H, Hopkinsville KY Workman, ♂ M.rubra	18.27	18.45	30.62	Rubra
91	LLP H Hopkinsville KY , Roberts Farm #6, ♀ M.rubra	18.92	18.92	31.18	Rubra
92	LLP H Hopkinsville KY , Roberts Mailbox, ♀ M.rubra	19.2	19.2	31.94	Rubra
93	LLP H Hopkinsville KY Harmony Grove (West), ♀ M.rubra	18.98	18.93	31.49	Rubra
94	LLP H, Hopkinsville KY 42240, Spillway, ♀ M.rubra	20.35	19.28	31.58	Rubra
95	Miles George, Aberdeen NC	21.35	30.7	20.94	Hybrid
96	David Barker, Scott LA	19.66	19.44	33.1	Rubra
97	Lara Plumb, Evansville AR , Tree #1	19.3	18.81	31.68	Rubra
98	Lara Plumb, Evansville AR , Tree #2	20.62	19.2	32.65	Rubra
99	Lara Plumb, Evansville AR , Tree #3	19.8	19.42	33.05	Rubra
100	Robert Gustafson, Machipongo VA #01, Male tree by Bay	20.19	19.59	33.29	Rubra
101	Robert Gustafson, Machipongo VA #02, Female tree, Large sharon's woods	19.16	19	29.58	Rubra
102	Robert Gustafson, Machipongo VA #03, Female, Small sharon's woods	19.07	19.08	30.63	Rubra

Discussion:
As expected the limited seedling trees that we acquired from nurseries advertised as Morus rubra ended up being hybrids or albas. This is not to say that native red mulberry cannot be found at domestic nurseries, but highlights the need for more education when collecting seed and a reminder that red fruit does not indicate a red mulberry tree. Given a visual guide and with experience of mulberries, many amateur fruit explorers were able to find native mulberries growing throughout the country, indicating that many more likely exist to be discovered and shared. Please find resources at https://thenakedoaktreefarm.com/red-mulberry for identifying trees yourself using a microscope or for fee-for-service testing of your collected specimens.

Please check back periodically to this report for updates on where to source red mulberry seed or cuttings, or email westonlombard@gmail.com until a resource becomes available.

Thank you to everyone who contributed to this exciting project. Looking forward to next steps.

Update re: Objectives #2 and #3:

Since the test results just came in, I wasn't able to create a reference guide for red mulberry cultivars yet, but am reaching out to people to see if they want their specimens and contact info listed and will eventually share a resource guide to help people access trees and cuttings. Might be a few months though.

As for the orchard preserves, we had planned to plant at Hocking College and at UVA Wise where there are not a bunch of established white mulberries already, but since they both dropped out of the grant, we had to plant at my farm. Instead of a pure rubra orchard, I have had to plant rubra's into my existing orchards wherever I could fit them and it still serves the purpose of a preserve, but isn't going to be able to work as a breeding area for pure morus rubra, because of how many alba and hybrids I have growing already. Still it is an area where people can see different specimens, sample diverse mulberry flavors, and collect scionwood.

Participation Summary

60 Farmers participating in research

Educational & Outreach Activities

3 Curricula, factsheets or educational tools

Participation Summary:

168 Farmers participated

5 Ag professionals participated

Education/outreach description:

In order to spread the word about our project and the Mulberry Search and Rescue Contest to land owners, farmers, and fruit enthusiasts, we first networked and consulted with other mulberry enthusiasts in order to create Contest Instructions and a resource list of Morus Rubra Identification Guides. Once this was in place we launched a Facebook Event with an easily shareable link that we could all distribute to our networks. This event link and the contest instructions were then shared on fruit growing forums, Facebook interest groups (primarily NAFEX and my own Growing Mulberry in Temperate Climates pages, and other targeted interest groups), and in email chains. The Facebook event and interest group pages were then regularly updated with videos about mulberry harvests, taste comparisons, and photos of fruit size. Because a really comprehensive identification video comparing the common mulberry species and hybrids wasn't accessible, we also created the most in-depth identification video available online.

We have now hosted a second round of the contest and collected an additional 50 samples. We are still working to complete the testing and share the results through similar avenues as well as direct messages to nurseries that may be interested in growing the newly released cultivars.

Learning Outcomes

5 Farmers reported changes in knowledge, attitudes, skills and/or awareness as a result of their participation

Lessons Learned:

We did confirm that thanks to the internet and the abundance of interest groups on specific topics like growing mulberries, that crowd-sourcing is a great way to find new trees worthy of propagation and sharing. This method has been used since at least the 1900's when people like John Hershey would put out ads in local papers requesting superior nuts and fruits be sent to him for evaluation. Thanks to the internet, this process is now much easier and cost effective and superior cultivars of any wild or domestic crop could be discovered and shared in such a manner.

The greatest problem we have faced is unique to this species and to figuring out the perfect and yet affordable genetic testing protocol for Morus rubra. Consulting with more geneticists prior to launch definitely would have helped for a smoother roll out of the project.

Project Outcomes

15 Farmers changed or adopted a practice

1 Grant received that built upon this project

5 New working collaborations

Information Products

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Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and should not be construed to represent any official USDA or U.S. Government determination or policy.