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Citation: Mayton, H.; Amirkhani, M.;
Loos, M.; Johnson, B.; Fike, J.;
Johnson, C.; Myers, K.; Starr, J.;
Bergstrom, G.C.; Taylor, A.
Evaluation of Industrial Hemp Seed
Treatments for Management of
Damping-Off for Enhanced Stand
Establishment. Agriculture 2022,12,
591. https://doi.org/10.3390/
agriculture12050591
Academic Editor: Giuseppe Lima
Received: 31 March 2022
Accepted: 21 April 2022
Published: 23 April 2022
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agriculture
Article
Evaluation of Industrial Hemp Seed Treatments for Management
of Damping-Off for Enhanced Stand Establishment
Hilary Mayton 1, Masoume Amirkhani 1, * , Michael Loos 1, Burton Johnson 2, John Fike 3, Chuck Johnson 4,
Kevin Myers 5, Jennifer Starr 5, Gary C. Bergstrom 5and Alan Taylor 1,*
1Cornell AgriTech, School of Integrative Plant Science, Horticulture Section, Cornell University,
Geneva, NY 14456, USA; hsm1@cornell.edu (H.M.); mtl72@cornell.edu (M.L.)
2Department of Plant Sciences, The College of Agriculture, Food Systems, and Natural Resources,
North Dakota State University, Fargo, ND 58108, USA; burton.johnson@ndsu.edu
3School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA 24061, USA; jfike@vt.edu
4Southern Piedmont Agricultural Research and Extension Center, Virginia Tech, Blackstone, VA 23824, USA;
spcdis@vt.edu
5School of Integrative Plant Science, Plant Pathology and Plant-Microbe Biology Section, Cornell University,
Ithaca, NY 14853, USA; klm25@cornell.edu (K.M.); jennifer.starr@iff.com (J.S.); gcb3@cornell.edu (G.C.B.)
*Correspondence: ma862@cornell.edu (M.A.); agt1@cornell.edu (A.T.)
Abstract:
The purpose of this research was to collect efficacy data on biological, biochemical, and
chemical fungicide seed treatments on hemp (Cannabis sativa L.) to mitigate damping-off and en-
hance field stand establishment. Seed treatments were evaluated in fields in New York (NY),
North Dakota (ND)
, and Virginia (VA) and at two planting dates in each state in 2020. A single
seed lot of a dual-purpose (fiber + grain) cultivar (‘Anka’) was treated using a laboratory-scale rotary
pan coater. Five biological, two biochemical, and four chemical seed treatments were tested. A labo-
ratory germination test revealed that seed treatments did not exhibit phytotoxicity when compared
to the non-treated control. A laboratory bioassay with naturally infested soil was used to assess the
preliminary activity of seed treatments for protection against damping-off. The biochemical seed
treatment Ultim
®
(active ingredient; organic copper) performed as well as the chemical treatments
Apron XL
®
+ Maxim
®
4FS and Mertect
®
340F in preventing damping-off whereas the biological
treatments did not differ from the non-treated control in terms of disease incidence. In all field tests,
biological seed treatments did not improve plant stands compared to the non-treated control. Bio-
chemical seed treatments Prudent 44
®
with Nutrol
®
(active ingredient; phosphite) and Ultim
®
, along
with chemical seed treatments, had acceptable efficacy and improved stand establishment compared
to the non-treated control across field locations. Based on efficacy results from laboratory and field
trials, the copper seed treatment has potential for both conventional and organic hemp production.
Keywords: biopesticides; biological control; Pythium;Cannabis sativa L.; organic copper; phosphite
1. Introduction
There is a renewed interest in the commercial production of hemp (Cannabis sativa L.)
in the United States (US) following the passage of the 2018 US Farm Bill that removed
the crop from Schedule I of the Controlled Substances Act [
1
,
2
]. C. sativa L. is most often
associated with its production of psychoactive terpenophenolic cannabinoids, specifically,
∆
-9-tetrahydrocannabiol (THC) [
3
,
4
]. However, industrial hemp with <0.3% THC is grown
for fiber, grain, and hempseed oil or for use as a nutraceutical primarily for cannabidiol
(CBD) production [
2
,
3
,
5
]. Hemp grown for grain and fiber is normally direct-seeded in
the field, whereas cannabinoid hemp is often grown in a greenhouse or transplanted to
the field [
6
]. Several recent publications reported numerous pest and disease problems
associated with the production of hemp [
7
–
9
]. Damping-off, along with powdery mildew,
Botrytis grey mold, and multiple leaf spot diseases, were all cited as impediments to
Agriculture 2022,12, 591. https://doi.org/10.3390/agriculture12050591 https://www.mdpi.com/journal/agriculture
Agriculture 2022,12, 591 2 of 12
commercial hemp production [
9
–
13
]. In addition, seedlings started in the greenhouse often
have poor root development that leads to increased susceptibility to pathogens and biotic
stresses when transplanted into the field.
Damping-off is specifically identified as a major problem in both field and green-
house production in the US and Canada [
8
,
14
–
17
]. The primary pathogens responsible for
damping-off in several studies were identified as Pythium sp., Fusarium sp., and Rhizocto-
nia [
8
,
14
,
16
,
17
]. The US restrictions placed on hemp in the 1930s limited research and no
pesticides were developed for disease control. Seed treatments are urgently needed for
efficient early season pest management, including the control of damping-off caused by soil
associated pathogens. Currently, pelleting, film coating, and encrusting are coating tech-
nologies used in the seed industry as delivery systems for plant protectants [
18
]. However,
the hard, waxy surface of hemp seeds necessitates film coating formulations for uniform
seed treatment application. Organically approved treatments would be ideal so that they
could be used in both conventional and organic production systems. Significant advances
were made in recent decades in the application of biopesticides as seed treatments that
have resulted in increased efficacy and more consistent disease control [19].
Biopesticides comprise two main categories, namely microbial-based products that
are either composed of microorganisms and the second category, which are biochemicals.
Biochemical products include minerals, plant and microbial extracts, enzymes, and organic
acids [
20
]. Several studies showed that biological seed treatments (BCA) that use Tricho-
derma and Bacillus species alone or in combination suppressed disease development and
improved plant growth [
21
–
23
]. Corn (Zea mays) seed coated with Trichoderma atroviride
had greater emergence and efficacy against two Fusarium pathogens in greenhouse and
field studies, and Trichoderma applications also enhanced plant defense and germination
when applied to cucumbers (Cucumis sativus L.) [
24
,
25
]. Zaim et al. [
26
] demonstrated
that a combination of B. subtilis and T. harzianum applied as seed treatments on chick-
pea suppressed disease development caused by F. oxysporum f. sp. ciceris by 93% and
enhanced plant growth. A combination of B. subtilis and T. asperellum applied as seed
treatments on tomato were effective in controlling Pythium aphanidermatum [
23
]. Biological
control of damping-off with a commercial formulation of Clonostachys rosea applied as a
combination of seed treatment and soil drench was as effective as mefenoxam in reducing
disease in seedlings of American ginseng (Panax quinquefolius) [
27
]. However, a commercial
formulation of Trichoderma harzianum was ineffective in this same study.
Biochemical seed treatments may be derived from minerals and elements, including
copper and phosphorus. Phosphites are widely studied in agriculture, and phosphites
as an alternative for the management of plant disease were reviewed in [
28
,
29
]. Phos-
phites, also known as phosphonates, are derived from phosphorus acid (H
3
PO
3
) and are
commonly combined with potassium, sodium, calcium, or ammonia. Phosphites exhibit
systemic movement in the plant and move in both the xylem and phloem. Phosphites have
demonstrated efficacy for a broad range of plant pathogens (oomycetes, fungi, bacteria, and
nematodes) in studies conducted on specific crops and applied with different application
technologies. Inorganic copper compounds were used for over a century as seed treatments
for the control of plant pathogens, and this was reviewed by Leukel (1936) [
30
]. The early
copper seed treatments were composed of copper sulfate, copper carbonate, or copper
hydroxide and formulated as dusts. Because inorganic copper treatments could cause
seed injury (phytotoxicity), they were largely replaced with synthetic fungicides [
31
]. The
chemical synthetic seed treatment fungicides mefenoxam and fludioxonil are labeled on
a wide range of crops [
32
,
33
]. Mefenoxam is effective against Pythium, while fludioxonil
controls soil-borne fungi, including Fusarium and Rhizoctonia. The efficacy of these chemical
seed treatments has not been reported on hemp.
Currently, there are neither registered organic controls nor chemical pesticides ap-
proved for use on hemp in the US. The purpose of this research was to evaluate a number
of biological, biochemical, and chemical seed treatments for the management of damping-
Agriculture 2022,12, 591 3 of 12
off caused by several pathogens (Pythium,Fusarium, and Rhizoctonia) to enhance stand
establishment.
2. Materials and Methods
2.1. Seed, Seed Treatments, and Laboratory Seed Treatment Testing
Seed of a single hemp seed lot (cv’Anka’, a monoecious dual purpose-fiber and grain
variety) was acquired from UniSeeds, Cobden, ON, Canada. The seed lot was first sized by
seed width to remove small seeds (<7.5/64 inch, or <3 mm). Seeds were treated at Cornell
AgriTech, Geneva, NY, USA, using a laboratory-scale rotary pan coater, R-6 (Universal
Coating Systems, Independence, OR, USA). Seed treatments and active ingredients are
listed in Table 1. The 20CU_2697LQ from ABM (now Agrauxine Corp.), Van Wert, OH,
USA, contained T. atroviride at a concentration of 2.6
×
10
9
colony forming units (cfu)/mL.
Amplitude
TM
,B. amyloliquefaciens strain F727, containing 1.0
×
10
9
cfu/mL, was provided
by Marrone Bio Innovations, Davis, CA, USA. Bio Seed contained five biologicals at
1.0 ×108
cfu for each species and was provided by Ag Biotech, Lakeville, NY, USA. Phyter
from Endo Plant Health, Oakville, ON, Canada, consisted of C. rosea at 1.0
×
10
9
cfu/mL.
Varnimo contained 1.0
×
10
9
B. amyloliquefaciens cfu/g, and KaPre Embrella, Prudent 44
®
and Nutrol
®
were provided by LidoChem Inc, Hazlet, NJ. Ultim
®
, a copper hydroxide
formulation, was obtained from Germains, Gilroy, CA, USA. All chemical fungicides
were acquired from Syngenta, Greensboro, NC, USA. EnVigor, an organic-based, multi-
functional seed coating polymer from Agrauxine, was used as the seed treatment binder
for all treatments except for the Ultim®seed treatment.
Table 1.
Seed treatments were applied to the Anka hemp seed lot with a laboratory-scale rotary
pan coater.
Treatment # Product Active Ingredient Rate/100 g Seed
1 Control - -
2 20CU_2697LQ T. atroviride 426 µL
3AmplitudeTM B. amyloliquifaciens strain F727 250 mg
4Bio SeedTM Paenibacillus azotofixans,B. megaterium,
B. mucilaginosus,B. subtilis,T. harzianum 400 mg
5 * Varnimo/KaPre Embrella B. amyloliquifaciens 200 + 2400 mg
6 Phyter Clonostachys rosea 50 mg
7Ultim®Copper hydroxide 0.05 mg Cu/seed
8Prudent 44®/Nutrol®Phosphite material +
Monopotassium phosphate 640 mg + 80 mg
9Apron XL®/Maxim®4FS 1/2X Mefenoxam + Fludioxonil 1
/2X rate
10 ** Apron XL®/Maxim®4FS 1X Mefenoxam + Fludioxonil 1 X rate
11 Apron XL®/Maxim 4FS®2X Mefenoxam + Fludioxonil 2 X rate
12 *** Apron XL®/Maxim®4FS
/Mertect®340F 1X Mefenoxam + Fludioxonil + Thiabendazole 1 X rate
for all products
* KaPre Embrella contains monosaccharides, disaccharides and oligosaccharides and natural compounds for
microbes. ** The 1X rate of Apron XL
®
/Maxim
®
4FS is 23.2 mg, 6.3 mg, and 11.8 mg/100 g, respectively. *** The
1X rate of corresponding active ingredients of Mefenoxam/Fludioxonil/Thiabendazole is 7.5 mg, 2.5 mg, and
5.0 mg/100 g, respectively.
A preliminary roll-towel soil bioassay was conducted to test the germination and
efficacy of the seed treatments. Field soil (Arkport fine sandy loam) was obtained from an
East Ithaca, NY, field location and contained the damping-off pathogen P. aphanidermatum
(Figure 1). The soil media used for the bioassay was composed of (by weight) 10% of the
field soil thoroughly mixed with 90% sand. Twenty-five seeds were placed linearly, 6 cm
Agriculture 2022,12, 591 4 of 12
from the edge of the moistened towels. A straight-edge shield was placed 4 cm from the
edge of the towel, allowing the seeds to be covered with 2 cm of soil, simulating in-furrow
planting depth. Using 100 g of the prepared soil media and a #10 sieve, the soil was evenly
shaken over the seeds and towel surface, then covered with a third towel and folded. Four
replicates were prepared using a roll-towel protocol [
34
], and germination results were
scored on days 4 and 7 with a final count on day 10 (DAP) (Figure 1).
Agriculture 2022, 12, x FOR PEER REVIEW 4 of 13
(Figure 1). The soil media used for the bioassay was composed of (by weight) 10% of the
field soil thoroughly mixed with 90% sand. Twenty-five seeds were placed linearly, 6 cm
from the edge of the moistened towels. A straight-edge shield was placed 4 cm from the
edge of the towel, allowing the seeds to be covered with 2 cm of soil, simulating in-furrow
planting depth. Using 100 g of the prepared soil media and a #10 sieve, the soil was evenly
shaken over the seeds and towel surface, then covered with a third towel and folded. Four
replicates were prepared using a roll-towel protocol [34], and germination results were
scored on days 4 and 7 with a final count on day 10 (DAP) (Figure 1).
Germination test criteria were adopted from the Association of Official Seed Analysts
(AOSA) manual [34], and germination was counted as a positive score when the seedling
radical was at least 2mm in length. For final counts, seedlings were only considered viable
when they met AOSA standards of healthy roots, hypocotyl, epicotyl, the presence of at
least ½ of cotyledon tissue, and no disease presence. Seedlings were scored as normal,
abnormal, or dead. Seeds were germinated in a Percival germinator (Percival Scientific,
Inc., model I36LL, Perry, IA, USA) set at 15/25 °C, night 10 h, day 14 h, with light provided
during the day period. The alternating 15/25 °C was selected based on the average diurnal
soil temperatures on June 1st in Geneva, NY, USA [35]. In addition, a rolled towel germi-
nation test was conducted without soil to assess seed treatment phytotoxicity. Seedlings
were scored as normal, abnormal, or dead after 7 days, and the germination test was also
conducted in the same Percival germinator at 15/25 °C, night 14 h, day 10 h, with light
provided during the day period.
4 DAP 7 DAP 10 DAP
Figure 1. Soil bioassay naturally infested with Pythium aphanidermatum. Ratings were recorded 4, 7
and 10 days after planting (DAP).
2.2. Field Trials
Trial locations, planting dates, and the dates of final seedling counts were recorded,
and soil types are shown in Tables 2 and 3. All planting sites were tilled prior to planting.
In NY, 100 seeds were sown per plot with four replications for both trials with a push
planter. The first trial in NY was planted on 12 June. The soil was an Arkport fine sandy
loam. The second trial in NY (Hudson/Collamer silt loam soil) was sown on 26 June. North
Dakota trials were planted on 20 June and 29 July into fine and firm seedbeds with a
Bearden/Perella silty clay loam soil. The field was conventionally prepared for a small-
seeded crop. Seeding in ND was performed using a 6-row (spaced at 30 cm), 3-point
mounted planter with double disk openers with twin-vee packer wheels. The center four-
planter rows were sown with 100 seeds placed at a 20 mm depth with a row length of 3.05
m. The first site in VA (Dothan/Norfolk sandy loam soil), planted at the Southern Pied-
mont Agricultural Research and Extension Center, was established with a forage seeder
on 10 April. Four replications were planted on each experimental plot, each plot contain-
ing two 6.1 m-long rows containing 100 seeds per row.
The second trial was also seeded
with four replications of 100 seeds in VA (Duffield/Ernst complex fine loam soil) and was
planted with a walk-behind cone seeder adapted for two rows. The second site was seeded
Figure 1.
Soil bioassay naturally infested with Pythium aphanidermatum. Ratings were recorded 4, 7
and 10 days after planting (DAP).
Germination test criteria were adopted from the Association of Official Seed Analysts
(AOSA) manual [
34
], and germination was counted as a positive score when the seedling
radical was at least 2mm in length. For final counts, seedlings were only considered viable
when they met AOSA standards of healthy roots, hypocotyl, epicotyl, the presence of at least
1
2
of cotyledon tissue, and no disease presence. Seedlings were scored as normal, abnormal,
or dead. Seeds were germinated in a Percival germinator (
Percival Scientific, Inc.,
model
I36LL, Perry, IA, USA) set at 15/25
◦
C, night 10 h, day 14 h, with light provided during
the day period. The alternating 15/25
◦
C was selected based on the average diurnal soil
temperatures on June 1st in Geneva, NY, USA [
35
]. In addition, a rolled towel germination
test was conducted without soil to assess seed treatment phytotoxicity. Seedlings were
scored as normal, abnormal, or dead after 7 days, and the germination test was also
conducted in the same Percival germinator at 15/25
◦
C, night 14 h, day 10 h, with light
provided during the day period.
2.2. Field Trials
Trial locations, planting dates, and the dates of final seedling counts were recorded,
and soil types are shown in Tables 2and 3. All planting sites were tilled prior to planting.
In NY, 100 seeds were sown per plot with four replications for both trials with a push
planter. The first trial in NY was planted on 12 June. The soil was an Arkport fine sandy
loam. The second trial in NY (Hudson/Collamer silt loam soil) was sown on 26 June.
North Dakota trials were planted on 20 June and 29 July into fine and firm seedbeds
with a Bearden/Perella silty clay loam soil. The field was conventionally prepared for
a small-seeded crop. Seeding in ND was performed using a 6-row (spaced at 30 cm),
3-point mounted planter with double disk openers with twin-vee packer wheels. The
center four-planter rows were sown with 100 seeds placed at a 20 mm depth with a row
length of 3.05 m. The first site in VA (Dothan/Norfolk sandy loam soil), planted at the
Southern Piedmont Agricultural Research and Extension Center, was established with a
forage seeder on 10 April. Four replications were planted on each experimental plot, each
plot containing two 6.1 m-long rows containing 100 seeds per row. The second trial was
also seeded with four replications of 100 seeds in VA (Duffield/Ernst complex fine loam
soil) and was planted with a walk-behind cone seeder adapted for two rows. The second
site was seeded on 4 October following several spring plantings that failed due to flooding.
The plots were tilled, and the soil was allowed to settle for a week before planting.
Agriculture 2022,12, 591 5 of 12
Table 2. Trial locations, planting and final stand count dates, and soil types at each location.
Trial Location Planting Dates Final Stand Counts (* DAP) Soil Type
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
New York 12 June 26 June 21 21 Arkport fine sandy loam Hudson/Collamer silt loam
North Dakota 20 June 29 July 27 27 Bearden/Perella silty clay loam Bearden/Perella silty clay loam
Virginia 10 April 4 Oct 25 20 Dothan/Norfolk sandy loam Duffield/Ernst complex fine loam
* DAP (days after planting).
Table 3. Trial GPS locations, and addresses.
Trial Location GPS Address
Trial 1 Trial 2 Trial 1 Trial 2
New York 42◦26027.70 0 N
76◦28015.00 0 W
42◦2702.2680 0 N
76◦26052.3420 0 WEast Ithaca, NY Dryden, NY
North Dakota 47◦00011.3000 N
97◦06041.950 0 W
47◦00011.300 0 N
97◦06041.950 0WCasselton, ND Casselton, ND
Virginia 37◦05034.8600 N
77◦57052.270 0 W
37◦1300.3650 0 N
80◦27045.7610 0 WBlackstone, VA Blacksburg, VA
Stand counts were recorded starting at day 7 and continued every 5–7 days for
3–4 week
s at each location. Stand counts were based on the above ground emergence
of the cotyledons and used to estimate the percentage emergence for each measurement
event. In addition, plants were evaluated for disease incidence via visual inspection for
signs and symptoms of pathogen development. After final counts, samples were destruc-
tively harvested at some locations and examined for root rot. Samples with signs of disease
were evaluated for pathogens at each location.
2.3. Field Pathogen Diagnosis
Diseased seedlings from field trials in Ithaca, NY, were collected while conducting
stand counts and taken to the Bergstrom laboratory located at Cornell University for identi-
fication. Samples were surface sterilized for 1 min each in 70% ethanol, 90% hypochlorite,
and sterile distilled water. Tissue was then plated on potato dextrose agar (PDA) with the
addition of streptomycin and neomycin and an oomycete selective agar (combination of
pea and rye B agar) [
36
]. Plates were incubated at room temperature with a light cycle of
12 h of UV-light and 12 h of darkness for 3–5 days until colonies could be isolated. Once
isolated into a pure culture, samples were identified morphologically and via sequencing
of the ITS rDNA region. Genomic DNA was extracted using a DNeasy Plant Mini Kit
(Qiagen), followed by PCR amplification of the ITS rDNA region using the universal ITS
primers ITS1 and ITS4 and the PCR method of White et al., 1990 [
37
]. The PCR fragments
were purified using a Monarch PCR and DNA clean-up kit (New England Biolabs), and
Sanger sequenced using Big Dye Terminator Cycle Sequencing and Clean-up Kits (Applied
Biosystems, Inc., Waltham, MA, USA), followed by analysis on an ABI 3730XL DNA an-
alyzer. The resulting sequence data were further processed using GeneStudio Sequence
Analysis Software (GeneStudio, Inc., Suwanee, GA, USA), followed by sequence best match
identification using NCBI BLAST [38].
Seedlings from North Dakota trials were carefully washed to remove soil that was at-
tached to the roots. Entire plants were examined with a dissecting microscope
(80
×
magnification) to observe general symptoms such as lack of roots, injuries, and
discolored tissue. When a pathogen was suspected, tissues (roots and stems were cut
lengthwise, leaves were cut into small pieces) were placed on temporary wet mounts and
viewed on a compound microscope (400
×
magnification). Slides were examined for the
presence of fungal structures (hyphae, spores) and bacterial streaming. Suspect arthropod-
pest afflicted tissues were examined by an entomological diagnostician to confirm the cause.
No disease was observed in trials located in Virginia.
Agriculture 2022,12, 591 6 of 12
2.4. Statistical Analysis
All seedling data from the trials were evaluated for normality, homoscedasticity, and
goodness of fit, along with the generation of histograms of the distribution of residuals
and normal probability plots, using JMP Pro Version 15 SAS Institute Inc., Cary, NC, USA,
1989–2021
. All data (including a soil bioassay; Table 4) were subjected to arcsine square
root transformation and evaluated for normality, homoscedasticity, and goodness of fit
using the same procedures described above. Non-transformed percent emerged seedlings
data had normal distributions in all trials across locations, and transformation did not
improve normality or tests of goodness of fit. Therefore, non-transformed raw data were
used to determine statistical differences in the percentage of emerged seedlings. Analysis of
variance was assessed, and a comparison of means was conducted with Tukey’s procedure
using JMP Pro 15 at a significance level of α= 0.05.
Table 4.
Percent germination test results (Normal seedlings, Abnormal seedlings and Dead) and Soil
Bioassay results for percent viable seedlings (4 Day, 7 Day, 10 Day).
* Percent Germination Test * Soil Bioassay
# Treatment Normal Abnormal Dead 4 Day 7 Day 10 Day
1 Non-treated control 89 4 7 97 A 34 C 1 B
2 20CU_2697LQ 83 5 12 82 B 17 C 0 B
3 Amplitude 79 9 12 84 AB 19 C 8 B
4 Bio Seed 84 3 13 89 AB 21 C 0 B
5 Varnimo/KaPreEmbrella 86 5 9 85 AB 51 BC 7 B
6 Phyter 87 2 11 93 AB 31 C 0 B
7 Ultim 85 3 12 97 A 89 A 71 A
8 Prudent44 + Nutrol 79 5 16 83 AB 49 BC 8 B
9 Apron XL + Maxim 4FS 1/2X rate 84 8 8 90 AB 80 A 65 A
10 Apron XL + Maxim 4FS 1X rate 85 5 10 94 AB 85 A 67 A
11 Apron XL + Maxim 4FS 2X rate 81 9 10 83 AB 79 A 68 A
12 Apron XL + Maxim 4FS +
Mertect 340F 1X 81 8 11 87 AB 71 AB 60 A
p≤value 0.2 NS 0.08 NS 0.5 NS 0.0023 **
<0.0001 ** <0.0001 **
* Arcsine data transformation was performed prior to ANOVA (Tukey HSD). NS—Not significant at 0.05. Mean
values with uncommon letters are statistically different. ** Significant at 0.01.
3. Results
3.1. Seed, Seed Treatments, and Laboratory Seed Treatment Testing
Normal, abnormal, and dead seedlings were counted in roll towel germination tests.
No differences in any germination parameters were observed for biological, biochemical,
and chemical seed treatments (#9–12, Tables 1,4and 5) when compared to the non-treated
control (Table 4). Results from the soil bioassay showed that only Ultim, an organic copper
treatment and chemical seed treatments (Apron XL + Maxim 4FS and in combination with
Mertect 340 F [rates presented in Tables 1,4and 5]) reduced damping-off compared to the
non-treated control and other treatments after 7 and 10 days. Varnimo and Prudent + Nutrol
showed some efficacy after 7 days; however, after 10 days, they were not significantly better
than the non-treated control.
Agriculture 2022,12, 591 7 of 12
Table 5.
Final stand counts (% emerged seedlings/plants) in two trials of hemp seed treatments
across three locations in 2020.
Location New York North Dakota Virginia
Trial Trial Trial
# Treatment 1 w2w1x2x1y2z
1 Non-treated control 33 C 27 37 CD 28 DE 14 CD 16
2 20CU_2697LQ 34 BC 32 36 CD 32 C-E 17 A-D 9
3 Amplitude 29 C 28 36 CDE 28 DE 15 BCD 13
4 Bioseed 31 C 30 33 D 34 B-D 12 D 10
5 Varnimo/KaPre Embrella 28 C 21 30 D 29 DE 16 BCD 22
6 Phyter 33 BC 24 40 C 26 E 14 CD 17
7 Ultim 42 A 35 49 B 42 A 21 A 28
8 Prudent 44 + Nutrol 41AB 29 38 C 27 DE 21 AB 17
9 Apron XL + Maxim 4FS 1/2X rate 32 C 22 58 A 43 A 20 ABC 17
10 Apron XL + Maxim4FS 1X rate 46 A 33 58 AB 40 AB 10 D 25
11 Apron XL + Maxim 4FS 2X rate 41 AB 42 56 AB 41 A 17 A-D 22
12 Apron XL + Maxim 4FS +
Mertect 340F 1X rate 30 C 28 55 AB 36 ABC 15 BCD 20
p≤value 0.0004 ** 0.09 NS 0.001 ** 0.0001 ** 0.05 * 0.12 NS
*, ** Significant at 0.05, and 0.01.
NS
—Not significant at 0.05. Mean values with uncommon letters are statistically
different. Final Count Days After Planting: w= 24 days, x= 27 days, y= 35 days, and z= 20 days.
3.2. Field Trials
Conditions were dry with above average air temperatures for the first trial in NY
planted on 12 June. The second trial in NY (Hudson/Collamer silt loam soil) was sown on
26 June and was again planted under dry conditions, followed by a hot and dry period.
Soil temperatures were greater than 16 and 21
◦
C for the North Dakota trials for the two
seeding dates, respectively, and rainfall 7 days post-planting was 9.6 mm and 0.0 mm for
seeding dates 20 June and 29 July, respectively. Although rainfall was minimal 7 days
post-planting, soil water status was adequate for seed germination for both seeding dates
due to rainfall occurring 7 days before planting.
Weather conditions were cool and damp at planting for the first site in VA, followed
by >5 cm of rain 24–30 h after planting. For the second trial in VA (Duffield/Ernst complex
fine loam soil), soil conditions were favorable, but seeding depth was inconsistent, and
there was little rainfall after planting.
Overall, the % of final emerged seedling counts measured for the two trials established
at different locations and planting dates (Tables 3–5) ranged from 9–58%. The highest
percentage of final emerged seedling counts were observed in ND and the lowest in VA
(Table 5). Trials in all three states had consistent seedling counts for both planting dates.
New York trials ranged from 28–46% and 21–41% in the first and second planting dates,
while ND ranged from 30–58% and 26–41%, respectively. Virginia with the lowest stand
counts ranged from 10–21% in the first trial and 9–25% in the second planting.
In five of the six seed treatment trials, most chemical seed treatments (#9–12,
Tables 1and 5
)
generally had higher stand counts than the biological seed treatments (#2–6, Tables 1and 5).
No differences were observed among seed treatments in the second trial conducted in VA.
Apron XL/Maxim 4FS applied at the 1X and 2X rates, and the biopesticides phosphite and
Ultim seed treatments had greater stand counts than the non-treated controls at E. Ithaca
(Table 5). At the second location in NY, only Apron XL + Maxim 4FS 2X rate had better
stands than the non-treated control (Table 5). The Ultim seed treatment (#7, Table 5) was
Agriculture 2022,12, 591 8 of 12
consistent across all locations and in most trials performed as well as the chemical seed
treatments (#9–12, Table 5).
3.3. Pathogen Diagnosis
Pathogens and/or diseased plants were observed and diagnosed in trials located in
NY and ND; however, no samples were collected from either VA trial. For trials in NY,
species of the damping-off pathogens Pythium,Fusarium, and Rhizoctonia were detected in
plants sampled at East Ithaca (Table 6and Figure 2). Due to dry conditions, no observations
of diseased roots and/or seedlings were obtained in the second NY trial. Rhizoctonia was
positively identified to species from samples treated with Amplitude in the first trial in
ND and also from the control, Apron XL Max
1
2
X rate, and Apron Max XL 2X rates in the
second trial. Ulocladium and Fusarium were found in the Bio Seed treatment and Apron
Max XL 1
2X rate plots in trial 1 in ND (Table 7).
Table 6. Sample and positive identification of pathogens present on hemp seedlings in Ithaca, NY.
Seed Treatment Pythium Species Fusarium Species Rhizoctonia Species Mucor Species Penicillium Species Trichoderma Species
Control x5x1x4x x
Amplitude (A) * x5x1x
Amplitude (B) * x5x1x2x4x x
Apron XL®/Maxim®
4FS 1/2X x1x2x3x4x
Bio Seed x5x1x2x3x4x
Varnimo/KaPre
Embrella (A) * x1x2x
Varnimo/
KaPreEmbrella (B) * x1x4
1
Fusarium equiseti
2
Fusarium solani
3
Fusarium oxysporum
4
Rhizoctonia solani
5
Pythium aphanidermatum * Letters
(A) and (B) indicate two separate samples were taken from the field plot I-3 (replicate I treatment 3).
Agriculture 2022, 12, x FOR PEER REVIEW 9 of 13
3.3. Pathogen Diagnosis
Pathogens and/or diseased plants were observed and diagnosed in trials located in
NY and ND; however, no samples were collected from either VA trial. For trials in NY,
species of the damping-off pathogens Pythium, Fusarium, and Rhizoctonia were detected in
plants sampled at East Ithaca (Table 6 and Figure 2). Due to dry conditions, no observa-
tions of diseased roots and/or seedlings were obtained in the second NY trial. Rhizoctonia
was positively identified to species from samples treated with Amplitude in the first trial
in ND and also from the control, Apron XL Max ½X rate, and Apron Max XL 2X rates in
the second trial. Ulocladium and Fusarium were found in the Bio Seed treatment and Apron
Max XL ½X rate plots in trial 1 in ND (Table 7).
Figure 2. Pathogens were observed on seedlings from treatments 1, 3, 4 and 5 and 9 (Tables 1 and
6). Damping-off pathogens were identified as P. aphanidermatum, F. equiseti, F. solani, F. oxysporum,
R. solani, Mucor, and Trichoderma species (Table 6) and the presence of bacterial species was also
identified on hemp seedlings from trial 1 in NY. Pathogen tests were conducted in the Bergstrom
lab, Cornell University.
Table 6. Sample and positive identification of pathogens present on hemp seedlings in Ithaca, NY.
Seed Treatment Pythium Species
F
usarium Species Rhizoctonia Species
M
ucor Species Penicillium SpeciesTrichoderma Species
Control x⁵ x¹ x4 x x
Amplitude (A) * x⁵ x¹ x
Amplitude (B) * x⁵ x¹ x² x4 x x
Apron XL®/Maxim® 4FS 1/2X x¹ x² x3 x
4 x
Bio Seed x⁵ x¹ x² x3 x
4 x
Varnimo/KaPre Embrella (A) * x¹ x² x
Varnimo/KaPreEmbrella (B) * x¹ x4
¹ Fusarium equiseti ² Fusarium solani 3 Fusarium oxysporum 4 Rhizoctonia solani ⁵ Pythium aphanidermatum
* Letters (A) and (B) indicate two separate samples were taken from the field plot I-3 (replicate I
treatment 3).
Table 7. Sample and positive identification of pathogens present on hemp seedlings in Prosper, ND.
Emergence Count Date
Seed Treatment 6 July 19 August
Control - Rhizoctonia
20CU_2697LQ - -
Amplitude Rhizoctonia -
Bio Seed Ulocladium -
Varnimo/KaPre Embrella - -
Phyter - -
Ultim - -
Figure 2.
Pathogens were observed on seedlings from treatments 1, 3, 4 and 5 and 9 (
Tables 1and 6
).
Damping-off pathogens were identified as P. aphanidermatum, F. equiseti, F. solani, F. oxysporum,
R. solani
,Mucor, and Trichoderma species (Table 6) and the presence of bacterial species was also
identified on hemp seedlings from trial 1 in NY. Pathogen tests were conducted in the Bergstrom lab,
Cornell University.
Table 7.
Sample and positive identification of pathogens present on hemp seedlings in Prosper, ND.
Emergence Count Date
Seed Treatment 6 July 19 August
Control - Rhizoctonia
20CU_2697LQ - -
Agriculture 2022,12, 591 9 of 12
Table 7. Cont.
Emergence Count Date
Seed Treatment 6 July 19 August
Amplitude Rhizoctonia -
Bio Seed Ulocladium -
Varnimo/KaPre Embrella - -
Phyter - -
Ultim - -
Prudent44/Nutrol - -
Apron XL/Maxim 4FS 1/2X Fusarium Rhizoctonia
Apron XL/Maxim 4FS 1X - -
Apron XL/Maxim 4FS 2X - Rhizoctonia
Apron XL/Maxim 4FS/Mertect 340F - -
Emergence count dates July 6 and August 19 correspond with planting dates June 20 and July 29, respectively.
(-) indicates no pathogens detected. Pathogen tests were conducted at the North Dakota State University Plant
Diagnostic Lab.
4. Discussion
High quality seed is the foundation for optimal stand establishment and the successful
production of any crop. Moreover, seed quality is especially important for hemp as a new
commercial crop with seed produced by a developing hemp seed industry. Currently, the
seed of most fiber and grain varieties are imported into the US, and seed lots may be of
low quality. Research at Cornell AgriTech’s Seed Science and Technology program tested
germination of 23 commercial hemp seed lots, and germination ranged from 29 to 94% in
2018 [
39
]. The minimum AOSCA standard for hemp is 80% germination [
40
], and only 13
of 23 lots tested in 2018 had greater than 80% germination. The germination of a hemp
seed lot can be improved by post-harvest seed conditioning [
41
]. Small-sized seeds of three
hemp varieties had a lower percentage of germination and produced seedlings with lower
dry weight than larger sized seeds [
42
]. Thus, removing the small-sized seeds effectively
upgraded seed lot quality. In this study, a commercial seed lot of ‘Anka’ was used, and
approximately 10% of the seed lot was removed, resulting in the non-treated control with
89% germination (Table 1).
There are several important factors and conditions needed for the efficacy of biologi-
cal control agents (BCA), starting with proper formulation and seed coating application
technology [
43
,
44
]. The BCA used in this investigation were from commercial sources, so
our assumption was that all the BCA were properly formulated. Envigor, a commercial
seed treatment binder, was tested in preliminary experiments and the CFU (colony forming
units) recovered from hemp seeds treated with a commercial mixture of Trichoderma spp. or
Bacillus amyloliquefaciens remained high. Therefore, the lack of efficacy was not attributed
to BCA formulation or seed coating technology. The environment in the soil bioassay
created severe disease pressure resulting in <10% seedling survival after 10 days (Table 4).
There are several mechanisms responsible for disease management in BCA, including
mycoparasitism [
45
]. The biocontrol organism may need to achieve a critical population
and produce its antifungal metabolites on roots in advance of pathogen growth in order to
provide protection to the plant or enhance plant defense. In addition, hemp seedlings may
be extremely susceptible to soil-borne pathogens, rendering these agents difficult to control.
Further investigation is warranted to explore BCA efficacy and mechanisms in relation to
damping-off on hemp seedlings.
Arguably, biochemical seed treatments have greater utility and flexibility than BCA
seed treatments as they do not contain living organisms that must remain viable during the
seed treatment process and post-treatment storage. Therefore, biochemical seed treatment
Agriculture 2022,12, 591 10 of 12
preparation, application, and storage are less complicated than BCA and thus more similar
to the application of chemical seed treatments [
18
]. A phosphite seed treatment, Prudent 44
in combination with Nutrol, was included in this study (Table 1). Prudent 44 contains 57%
by wt. urea phosphite and 15% by wt. ammonium phosphite [
46
]. Nutrol is monopotassium
phosphate and is registered with the US EPA (Environmental Protection Agency), and
Nutrol, when combined with Prudent, has expanded efficacy [
47
]. Since phosphites do not
exist in nature, formulations containing phosphites are not approved for organic labelling.
Prudent 44 with Nutrol provided some level of protection in the soil bioassay (Table 4) and
two (Table 5-Trial 1 of New York and Virginia) of the six field tests. A commercial phosphite
formulation (AG3) was applied as a seed soak, controlled damping-off caused by Pythium
spp. in Cucumis sativus [
48
]. Phosphite applied as a seed treatment to corn reduced the
mycelial growth of two Fusarium species [49].
Collectively, phosphites demonstrated potential as a hemp seed treatment, although
efficacy was generally less than copper or the chemical fungicide seed treatments. Ultim
treatment was performed as well as the chemical treatments at all locations and was the
only treatment that was not significantly different from the chemical treatments in the soil
bioassay (Tables 4and 5). Soil type and planting conditions significantly influenced hemp
stand establishment and growth; however, chemical seed treatments and Ultim were able
to improve overall stand establishment at most locations. Currently, the registrant for the
chemical seed treatments was not supportive of registering their materials for commercial
use on hemp. The active component in Ultim is copper hydroxide, a patented formulation
that is approved for organic use [
50
]. Collectively, the organic copper seed treatment has
potential for both conventional and organic hemp production. Moreover, results from this
study provide the foundation for seed treatment research on Cannabis sativa grown for
other uses. For example, Ultim, the organic copper treatment evaluated in this study, and
other organic copper seed treatments were found to be effective in controlling damping-off
in the soil bioassay on a CBD (cannabidiol) variety of hemp [51].
Author Contributions:
The first author H.M. contributed to the experimental design, data collection
and analysis, writing of the original draft, and revisions of the manuscript. The second author, M.A.,
assisted with planting field trials, data analysis, and preparation of the manuscript. The third author,
M.L., conducted laboratory bioassays and assisted with planting and data collection in the field. The
fourth, B.J., fifth, J.F., and sixth, C.J., authors were involved in the establishment and collection of data
for field trials conducted in ND and VA. The seventh, K.M., eighth, J.S., and ninth, G.C.B., authors
were responsible for plant pathogen identification for trials in NY. The last author, A.T., served as
the principal investigator and was involved with all aspects of experiments, including experimental
design and with the preparation and revisions of the manuscript. All authors have read and agreed
to the published version of the manuscript.
Funding:
This research was funded by the IR4 and NYS EDS 90512. This material is based upon
work that is supported by the United States Hatch Funds under Multi-state Project W-4168 under
accession number 1007938.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors thank company registrants for contributing their seed treatment
formulations and expertise in conducting this research: Agrauxine, Ag Biotech, Endo Plant Health,
Germains, LidoChem, Marrone Bio Innovations, and Syngenta. We also thank Incotec (Salinas, CA)
for seed treatment binders and film coating technology.
Conflicts of Interest: The authors declare no conflict of interest.
Agriculture 2022,12, 591 11 of 12
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