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Surveying for Potential Diseases and Abiotic Disorders of Industrial Hemp (Cannabis sativa) Production

    Affiliations
    Authors and Affiliations
    • Lindsey D. Thiessen1 †
    • Tyler Schappe1
    • Sarah Cochran1
    • Kristin Hicks2
    • Angela R. Post1
    1. 1North Carolina State University, Raleigh, NC 27603
    2. 2North Carolina Department of Agriculture, Agronomic Services Division, Raleigh, NC 27607
    Published Online:14 Oct 2020https://doi.org/10.1094/PHP-03-20-0017-RS

    Abstract

    Industrial hemp (Cannabis sativa L.) has recently been reintroduced as an agricultural commodity in the United States, and, through state-led pilot programs, growers and researchers have been investigating production strategies. Diseases and disorders of industrial hemp in the United States are largely unknowns because record-keeping and taxonomy have improved dramatically in the last several decades. In 2016, North Carolina launched a pilot program to investigate industrial hemp, and diseases and abiotic disorders were surveyed in 2017 and 2018. Producers, consultants, and agricultural extension agents submitted samples to the North Carolina Department of Agriculture and Consumer Services Agronomic Services Division (n = 572) and the North Carolina Plant Disease and Insect Clinic (n = 117). Common field diseases found included Fusarium foliar and flower blights (Fusarium graminearum), Fusarium wilt (Fusarium oxysporum), and Helminthosporium leaf spot (Exserohilum rostratum). Greenhouse diseases were primarily caused by Pythium spp. and Botrytis cinerea. Common environmental disorders were attributed to excessive rainfall flooding roots and poor root development of transplanted clones.

    Industrial Hemp Production in the United States

    Industrial hemp (Cannabis sativa L.) has had an important role in U.S. history. Hemp was an important textile in early settlements of the United States (Clark 1916), and production continued to be important through the 1800s (Clark 1916; Fortenbery and Bennett 2004; Kraenzel et al. 1998; Roulac 1997). Decreases in availability of labor and increased mechanization of other textiles reduced the production of hemp into the early 1900s (Clark 1916; Fortenbery and Bennett 2004; Kraenzel et al. 1998; Roulac 1997). Production was later controlled by the Marijuana Tax Act (U.S. Congress, House of Representatives 1937), which controlled cannabis production in the United States (Fortenbery and Bennett 2004). The need for fiber during World War II temporarily encouraged hemp production with the promotion of “Hemp for Victory,” but the production was reduced again after the war efforts were no longer needed (Fortenbery and Bennett 2004).

    Industrial hemp has again become legal to grow in the United States with state-led industrial hemp pilot programs and more recently with the Agriculture Improvement Act of 2018. These pilot programs and federal legalization have increased acreage of the crop. In the spring of 2017, North Carolina introduced an industrial hemp pilot program to assess the agronomic feasibility and economic impact of commercial production to the state. Although initially thought to be a potential fiber and seed market, North Carolina producers have primarily grown therapeutic (high-cannabidiol [CBD]) cannabis strains (North Carolina Department of Agriculture and Consumer Services [NCDA&CS]) during the pilot program. In 2017, about 100 growers were licensed by the state to produce industrial hemp, and production increased in 2018 with growers licensed in about 75% of counties within the state. This increased production has led to questions regarding potential causes for significant losses, especially those by pathogen pressures, and the legality of management practices (Stone 2014). Because the scope of plant pathogen impact is unknown, it is important to identify pathogen pressures.

    Agronomic management of industrial hemp is poorly understood in the United States, especially given the variety of uses for which industrial hemp is grown. Fiber- and grain-producing hemp are largely direct seeded, whereas industrial hemp for CBD production is transplant based. Much of the previous work on industrial hemp has focused on fiber and grain varieties (Dewey 1914; Ehrensing 1998; Meijer et al. 1995; Pickering et al. 2007; Ranalli and Venturi 2004; Sankari and Mela 1998; Van der Werf et al. 1995) compared with recreational (high tetrahydrocannabinol [THC]) and therapeutic varieties (Nissen et al. 2010). Industrial hemp was previously a textile crop in the southeast United States (Dewey 1914) and had relatively few abiotic and biotic stressors cited. Currently, the predominant hemp type grown in North Carolina is therapeutic hemp produced for CBD. Recreational and therapeutic hemp types, however, do not have documented pest pressures in the Southeast United States, which may limit the profitability of these crops in the region. Similarly, the nutritional needs of these hemp types are also poorly understood, which limits the ability for tissue and soil assays to be valuable for producers.

    The climate in the southeast United States, a humid subtropical climate (Cfa), is conducive for the development of many plant pathogens and is characterized by lacking distinct wet and dry seasons with at least 1 month averaging above 22°C and the coldest month averaging above 0°C (Kottek et al. 2006). The region receives an average of 1,016 to 1,397 mm of rainfall; however, the rainfall in 2018 was significantly greater than previous years, with many places up to 760 mm of precipitation above yearly averages (North Carolina Climate Office 2020). Because industrial hemp’s origins are thought to be within Central Asia in regions with less consistent moisture (Russo 2007), high precipitation may cause damage to plant tissues (McPartland et al. 2000). Significant moisture and warm conditions favor the growth of fungi, bacteria, and vectors for viruses that could significantly impact yield and quality of industrial hemp products. Gray mold (Botrytis cinerea), powdery mildew (Podosphaera macularis and Golovinomyces cichoracearum), and hemp canker (Sclerotinia sclerotiorum), among others, have been identified on C. sativa (McPartland et al. 2000), but recent publications identifying pathogens are few (McPartland and Cubeta 1997). This is likely due to the extensive gap in production of hemp from the early to mid-1900s to now. In addition to biotic pressures on industrial hemp production, abiotic disorders are relatively unknown for current strains of industrial hemp. The limited information on the abiotic and biotic pressures limits the ability to create cost-effective integrated management programs for industrial hemp.

    The primary objectives of this project was to (i) evaluate the diversity of pathogens of industrial hemp in the region and confirm the pathogenicity of the most economically important species found using Koch’s postulates, (ii) evaluate and confirm abiotic disorders and their causes found in the region, and (iii) determine grower perception of issues producing industrial hemp.

    Sampling for Hemp Risk

    Sample collection.

    Samples from commercial industrial hemp production were collected from submissions to the North Carolina State University Plant Disease and Insect Clinic (PDIC) in 2017 (n = 20) and 2018 (n = 98) or to the North Carolina Department of Agriculture Agronomic Services Division (ASD) in 2017 (n = 113) and 2018 (n = 459). Plant samples submitted to the PDIC included plant parts (leaves, roots, or stems) or whole plants that included soil. Fungal organisms were identified using morphological characteristics under compound and dissecting microscopes. Nonobligate plant pathogens were isolated onto media: fungi and oomycetes onto water agar and bacteria onto nutrient agar. Cultures were then maintained on potato dextrose agar (PDA) for further testing.

    Samples exhibiting symptoms consistent with abiotic stresses were submitted for additional evaluation by extension professionals or further testing including tissue analysis. Samples submitted for testing to the ASD included soil samples and leaf samples for nutrient assays. Tissue samples consisted of the most recent mature leaves collected from plants exhibiting symptoms.

    Molecular identification.

    Representative isolates established in pure cultures for each pathogen were used for molecular identification. Representative cultures were chosen based on morphological features and being free from contaminants. From fungal cultures, spores and hyphae were harvested by aliquoting 7 ml of sterile water into the Petri dish and gently scraping hyphae and spores from the surface with a sterile rubber spatula. The suspension was hand mixed, and 450 µl was added to the first step of the DNeasy PowerSoil DNA extraction kit (Qiagen, Hilden, Germany). For bacterial samples, a single bacterial colony was added to the first step of the DNeasy PowerSoil DNA extraction kit using a sterile bacterial loop. All remaining DNA extraction steps were consistent with manufacturer recommendations.

    A multilocus sequence typing approach was used to determine species identity of the unknown isolates with gene regions chosen depending on the known taxonomic group (fungi, bacteria, oomycetes). For fungi, at least two of the following loci were used: ITS1/ITS2 with primers ITS1f (Gardes and Bruns 1993) and ITS4 (White et al. 1990), DNA-directed RNA polymerase II subunit (RPB2) with primers bRPB2-6F and bRPB2-7R (Matheny 2005), beta-tubulin (TUB2) with primers T1 and T22 (O’Donnell and Cigelnik 1997), translation elongation factor 1-α (EF1-α) with primers EF1 and EF2 (O’Donnell et al. 1998), and 18S rRNA with primers nu-SSU-0817-5′-24 and nu-SSU-1647-3′ (Banos et al. 2018). Loci sequenced were chosen based on regions that are specific for identification of each fungal organism and adequate reference isolates in taxonomic databases for comparison. For oomycetes, the loci used were ITS1/ITS2 and cytochrome oxidase subunit I (COI) with primers COI-Lev-up and FM85 (Man in ’t Veld et al. 2015). For bacteria, the loci used were 16S rRNA with primers S-D-Bact-0008-a-S-16 and S-D-Bact-1492-a-A-16 (Muyzer et al. 1995) and ribosomal polymerase beta subunit (rpoB) with primers rpoB CM7-F and rpoB CM31b-R (Brady et al. 2008). Specific loci chosen for each organism are listed in Table 1. Each locus was amplified via polymerase chain reaction (PCR) using conditions found in the references for each primer set described above in 25-µl reactions with 12 µl of AccuStart II PCR ToughMix (2×), 2 µl of template DNA, 9 µl of nuclease-free water, and 1 µl of each primer pair (10 µM). PCR products were visualized via gel electrophoresis, cleaned up using AMPure XP magnetic beads, and sequenced in both directions using Sanger sequencing at the North Carolina State University Genomic Sciences Laboratory (Raleigh, NC). Forward and reverse raw sequences were quality trimmed, aligned using MAFFT 7.450 (Katoh and Standley 2013; Katoh et al. 2002), and a consensus sequence was generated based on the highest quality from the electropherogram peaks using Geneious Prime version 2019.2.1. Given the variable success of each primer set among isolates, not all isolates were able to be sequenced using all loci available (Table 1).

    TABLE 1 Diseases and causal agents collected from commercial industrial hemp samples submitted to the North Carolina State University Plant Disease and Insect Clinic in 2017 and 2018 (n = 20 and 98, respectively). All samples represented in this table were identified using a multilocus Sanger sequencing approach to identify bacteria, fungi, and oomycetes or species-specific primers designed for root-knot nematode identification

    To initially determine species identity, consensus sequences for each locus were then used to query the NCBI nucleotide database using BLASTn (Altschul et al. 1990) to identify sequences with the greatest similarity based on percent grade and to acquire taxonomic information. To ensure accurate taxonomic identification, phylogenetic trees were constructed for a subset of unknown isolates and reference sequences from NCBI GenBank. First, reference sequences of each locus for representative isolates of all species within the genus of each selected unknown isolate were downloaded from NCBI GenBank. Second, alignments were made using both unknown and reference sequences for each genus for each locus sequenced using MAFFT 7.450. Next, for each genus, alignments of different loci were concatenated together. Finally, best-scoring maximum-likelihood phylogenetic trees were constructed using RAxML 8 (Stamatakis) using the GTR GAMMA I model from 1,000 bootstrap replicates for each concatenated alignment for each genus.

    For root-knot nematode samples, females were removed directly from root tissues using a dissecting needle. To extract DNA, females were macerated in 50 µl of pH 8.0 1× Tris-EDTA (TE) buffer (Fisher Scientific, Waltham, MA) using a sterile plastic pestle. Immediately after maceration, TE-DNA extracts were used in PCR reactions specific to root-knot nematode species. Primers specific to Meloidogyne spp. were developed for sequence characterized amplified region (SCAR), including Meloidogyne enterolobii (Tigano et al. 2010), M. javanica, M. arenaria, M. incognita (Donkers-Venne et al. 2000), and M. hapla (Wishart et al. 2002). Reactions contained a total volume of 20 µl, with 1 µl of template DNA, 10 µM forward primers, 10 µM reverse primers, 10 µl of AccuStart II PCR Supermix (Quantabio, Beverly, MA), and 8 µl of molecular-grade water. All reactions were cycled according to the reaction conditions specified by the developer for each primer set (Donkers-Venne et al. 2000; Tigano et al. 2010; Wishart et al. 2002). Amplicons were visualized using electrophoresis through a 1% agarose gel and GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA) and compared with a 10-kb ladder standard.

    Koch’s postulates.

    Because the original hemp cultivar was not necessarily indicated by the producer when samples were submitted to the PDIC, one cultivar of hemp was used for Koch’s postulate testing. Industrial hemp ‘Carmagnola’ (HemPoint, Czech Republic) seedlings were produced by sprouting seeds in wax paper rolls that had one end submerged in a beaker of tap water. After 48 h, germinated seeds were planted in propagation mix consisting of 1/3 peat-lite and 2/3 gravel substrate (Sun Gro Horticulture, Agawam, MA). Plants were grown in controlled-environment incubators at 25°C with a 14-h photoperiod until two true leaves were produced by plants (approximately 3 weeks). Due to the large size reached by mature hemp plants, seedlings were used for pathogenicity assays to be evaluated in controlled environmental conditions.

    Spore suspensions of spore-producing fungal cultures were created by adding 7 ml of sterile deionized water to cultures and then gently scraping spores from hyphae using a sterile rubber spatula. Spores were filtered from hyphae by straining the suspension through four layers of cheesecloth. The spore suspension was then quantified and diluted or concentrated to 104 spores/ml. Hemp plants were inoculated with spore suspensions by spraying leaf surfaces until covered using a Preval hand sprayer (Nakoma Products, Bridgeview, IL). Nontreated controls were inoculated with sterile water. For nonsporulating fungi, hyphal plugs (7.3-mm diameter) were collected from the growing edge of PDA culture plates. Hyphal plugs were placed on hemp plant crowns or roots, hyphae-side to the plant, and then wrapped with Parafilm to hold the plug on the stems until ratings ceased. Nontreated controls were treated with a sterile medium plug similar to that described previously to mimic potential symptom development in the absence of a causal agent.

    Bacterial suspensions of collected bacterial pathogens were created by adding 7 ml of sterile deionized water to a culture plate and disturbing the culture with a rubber spatula. The optical density (OD) was then measured using a Thermo Scientific Spectronic 20D+ spectrophotometer (Waltham, MA), and suspensions were diluted to 0.01 OD. Hemp leaf bacteria were inoculated by spraying suspensions over the leaf surface until water collected on the leaf surface. Nontreated controls were inoculated with sterile water. Bacterial vascular diseases were inoculated by drenching soil with bacterial suspension and wounding roots by cutting with a sterile scalpel blade (Hong et al. 2012). Nontreated control plants were drenched with sterile water, and roots were wounded with a sterile scalpel blade.

    For each pathogen isolated from field samples, three plants were inoculated and three not inoculated. Plants were maintained in growth chambers and monitored three times a week for 3 weeks until disease symptoms were present. Once symptoms were present, plants were monitored daily until harvest. After disease development occurred, lesions were excised from plants using a sterile scalpel and then surface sterilized with a 10% bleach solution and allowed to air dry. Lesions were then plated onto water agar for fungi or nutrient agar for bacteria and allowed to grow for 48 h. Culture identity was confirmed by microscopic morphological identification of fungal structures and colony morphology of bacteria.

    Abiotic disorders.

    Abiotic samples were cataloged and imaged to assist in building a future comparative diagnostic guide for differentiating between industrial hemp diseases and common abiotic stresses. The ASD evaluated 113 samples in 2017 and 459 samples in 2018.

    For plant tissue analysis, samples were dried overnight (12 to 24 h) at 80°C and then processed through a stainless steel grinder (Wiley Mini-Mill; Thomas Scientific, Swedesboro, NJ) with a 20-mesh (1-mm) screen (Campbell and Plank 1992). Total nitrogen concentration (percent per gram weight of sample) was determined by oxygen combustion gas chromatography with an elemental analyzer (NA1500s2; CE Elantech Instruments, Lakewood, NJ) (AOAC 1990; Campbell 1992) on a 9- to 11-mg aliquot of the dried/ground sample.

    Total concentrations of P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, B, Na, and Al were determined with inductively coupled plasma optical emission spectrometry (Spectro Arcos EOP, Spectro Analytical: A Division of Ametek, Mahwah, NJ) (Donohue et al. 1992), after closed-vessel nitric acid (HNO3) digestion in a microwave digestion system (MARS 6 Microwaves; CEM Corp., Matthews, NC) (Campbell and Plank 1992). A 0.5-g dried/ground aliquot was microwave digested in 10 ml of 15.6 N HNO3 for 30 min, and the prepared sample volume was brought to 50 ml with deionized water and then filtered through acid-washed filter paper (Laboratory Filtration Group, Houston, TX) prior to measurement. Results for N, P, K, Ca, Mg, S, and Na are expressed as a percentage (%) and for Fe, Mn, Zn, Cu, B, and Al in parts per million (ppm) (as milligrams per kilogram [mg/kg]) on a dry-weight basis.

    Survey of hemp producers.

    In North Carolina, industrial hemp growers participating in the pilot program and holding a permit must complete an annual survey about their production practices and experiences. The NCDA&CS administers the survey each year of the pilot program in conjunction with North Carolina State University. For the 2018 growing season 344 producers responded to the survey, and of those 252 actually planted a crop in 2018. All data from the survey in this paper are reported as a percent of producers who actually produced the crop (n = 252). Producers were asked about their production practices, including variety planted, cultivation practices, weed control practices, and crop rotation. Growers were also asked about problems observed (abiotic and disease related), including diseases present, abiotic stress, and nutrient deficiencies or toxicities. Problems observed and self-reported were summarized for 2018 commercial production.

    Diseases Identified from Survey Collections

    PDIC survey.

    Samples submitted to the PDIC increased from the start of the North Carolina Industrial Hemp Pilot program in 2017 (n = 20) to 2018 (n = 97), many of which had multiple disease-causing agents on a single sample. Climatic conditions were different between 2017 and 2018, with significantly greater rainfall occurring in 2018 (151 cm in Raleigh, NC) compared with 2017 (114 cm in Raleigh, NC), with higher rainfall values observed along the coast (data not shown). Across both years, 16 diseases caused by fungi, bacteria, oomycetes, and nematodes were observed, with 10 being confirmed with Koch’s postulates and molecular identification accounting for 65 and 96% of samples collected in 2017 and 2018, respectively (Table 1). Of the total number of samples identified in the PDIC over 2017 and 2018, Fusarium foliar and flower blight (Fusarium graminearum) was most consistently prevalent across both years, accounting for 16% of sample diagnoses in 2017 and 20% in 2018 (Table 1). Helminthosporium leaf spot (Exserohilum rostratum) decreased from 16% of samples to 11% of samples (Table 1). All organisms identified in Table 1 were found to be pathogenic in Koch’s postulates assays, and lesion descriptions by organism are described below. Multilocus phylogenetic analyses for selected genera are presented in Figure 1.

    FIGURE 1

    FIGURE 1 Phylogenetic trees of representative pure isolates of unknown fungal samples from study and representative sequences from the NCBI nucleotide database. For a given putative genus that each unknown sample belongs to based on initial BLASTn results, representative sequences from all species within that genus were downloaded from NCBI for each locus. Letters represent trees from putative genera or taxonomic groupings: A, Botrytis; B, Exserohilum/Setosphaeria; C, Fusarium; D, Pythium; and E, Atheliaceae. The Atheliaceae phylogeny was expanded to the family level because not enough sequences were available for species within the Athelia genus. Sequences for each gene region were aligned separately using MAFFT version 7.388, and then alignments were concatenated and masked to remove all gaps. Phylogenetic trees were created using RAxML 8 using the GTR GAMMA I nucleotide model and rapid bootstrapping and search using 1,000 bootstrap replicates.

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    Gray mold.

    Gray mold (Botrytis cinerea) was identified on plants grown in greenhouse production (<2%) in fall of 2018 (Table 1), and it continues to be found in greenhouse production in the region (data not shown). Lesions were found on stems, petioles, leaves, and flowers, and significant necrosis was observed in the center of lesions. Lesions begin initially as small water-soaked lesions and then expand to entire plant parts, where abundant hyphae and sporulation are observed. Plants with stem lesions showed symptoms of leaf yellowing and wilting, and lesions contained fungal mats with abundant hyphae (Fig. 2A). Leaves, petioles, and flowers with lesions were necrotic with yellow margins on lesions, with abundant hyphae and sporulation in lesions (Fig. 2A and B). Conidiophores were tall and dark brown with irregular branching, and each conidiophore bore hyaline, single-celled, ovoid or globose conidia. Isolates produced irregularly shaped sclerotia (1 to 3 × 2 to 4 mm) in culture on the edge of culture plates. BLASTn queries of ITS1/ITS2 and RPB2 sequences found greatest similarity to Botrytis cinerea and Botryotinia fuckeliana, respectively, the latter being the teleomorph (Table 1). The multilocus phylogeny based on ITS1/ITS2 and RPB2 of the Botrytis genus showed that the representative unknown isolate grouped most closely with the Botrytis cinerea reference sequence with 90% bootstrap support (Fig. 1A).

    FIGURE 2

    FIGURE 2 Industrial hemp disease symptoms and signs observed during the 2017 and 2018 survey period. Gray mold caused by Botrytis cinerea hyphae and spores on decayed leaf (A) and petiole tissues (B). Leaf chlorosis (C), root necrosis (D), and oospores within roots (E) of Pythium spp. causing root rot. Hyphal growth on dried industrial hemp floral parts (F), green floral parts (G), leaf spots with darkened margins (H), and stem canker (I) caused by Fusarium spp. Wilting symptoms (J) and hyphal growth and sclerotia formation (K) on surface of root tissues.

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    Pythium root and crown rot.

    Pythium root and crown rot was observed on 3 and 8% of samples submitted from greenhouse production in all parts of the growing season, often in situations of overwatering or float-tray clone production, in 2017 and 2018, respectively (Table 1). Aboveground portions of plants showed symptoms of wilting and leaves with marginal yellowing and chlorosis (Fig. 2C). Roots of symptomatic plants were necrotic, with water-soaked lesions (Fig. 2D). Oospores were observed within symptomatic root tissues (Fig. 2E). Under light microscopy, morphological characteristics included spherical, terminal sporangia and intercalary sporangia, which were most consistent with those of Pythium aphanidermatum and Pythium ultimum (Beckerman et al. 2017, 2018; Watanabe et al. 2008). BLASTn queries of representative root and crown rot isolates using ITS1/ITS2 found the greatest similarity with Pythium ultimum and Pythium myriotylum, respectively. Queries using COI supported an identity of Pythium ultimum for the root rot isolate but for the crown rot isolate found greatest similarity with Pythium aphanidermatum (Table 1). In the multilocus phylogeny based on ITS1/ITS2 and COI (Fig. 1D), the root rot isolate grouped most closely with Pythium ultimum, but only with 22% bootstrap support. The next closest node also contained Pythium splendens and had 96% bootstrap support. The crown rot isolate grouped most closely with a clade containing Pythium myriotylum, Pythium zingiberis, and Pythium scleroteichum, but not with Pythium aphanidermatum. Taking the BLAST and phylogenies together, the root rot isolate was found to be Pythium ultimum, and the crown rot isolate was Pythium myriotylum.

    Bacterial leaf spot.

    Bacterial leaf spots were observed on 19 and 7% of plants from greenhouse production in all portions of the growing seasons of 2017 and 2018, respectively (Table 1). Lesions were caused by several bacteria, including Sphingobium yanoikuyae, Pseudomonas koreensis, and Serratia marcescens. Lesions caused by Sphingobium yanoikuyae and Pseudomonas koreensis were small (1 to 5 mm), medium brown to dark brown, angular, and limited to leaf veins. Serratia marcescens lesions were small (1 to 5 mm), dark brown to black, angular, and limited to leaf veins. Later, lesions caused by Serratia marcescens coalesced to large black lesions encompassing large areas of the leaf. Puncturing lesions caused by Serratia marcescens resulted in red bacterial ooze emerging from the lesion. As leaf disease caused by all bacterial pathogens progressed, lesions coalesced to form large necrotic lesions that encompassed large portions of the leaves. In culture, Sphingobium isolates produced yellow, convex colonies on nutrient agar, which could be easily confused with other plant pathogenic bacteria such as Xanthomonas spp. Pseudomonas isolates produced eggshell white, convex colonies on nutrient agar. Serratia marcescens cultures produced salmon-pink, convex colonies on nutrient agar (Schappe et al. 2019).

    Although not commonly implicated as a plant pathogen, Sphingobium yanoikuyae produced distinct lesions in pathogenicity assays on seedlings without wounding. Similarly, Pseudomonas syringae can persist in planta without causing disease; however, inoculations without wounding resulted in distinct lesions on leaf tissues. Lesion development after inoculations by Serratia marcescens were observed despite no physical wounding to plant tissues, as well, and lesions rapidly expanded across the leaf, creating large portions of necrotic tissues. Because lesions were observed with all inoculations in the absence of wounding, these organisms are speculated to invade plant tissues through hydathodes, trichomes, and/or stomata (Barak et al. 2011; Gu et al. 2013; Underwood et al. 2007).

    BLASTn queries based on 16S rRNA and/or rpoB showed that pure cultures of each representative bacterial isolate were most similar to Sphingobium yanoikuyae, Pseudomonas syringae, and Serratia marcescens (Schappe et al. 2019), respectively (Table 1). Although not previously identified as a pathogenic organism of hemp, Sphingobium yanoikuyae has been previously identified as pathogenic on Christ’s thorn (Paliurus spina-christi) (Deldavleh et al. 2013). This serves as a first report of Sphingobium yanoikuyae as a plant pathogen of hemp.

    Fusarium wilt.

    Fusarium wilt (Fusarium oxysporum) was found on 17% of samples collected from the field in 2018 during all parts of the growing season (Table 1). Plants were found with significant wilting and chlorosis of leaves. Upon cross-section, brown to pink-purple vascular discoloration was observed in stems. Fungi isolated from stems onto water agar then transferred to PDA had abundant white hyphae with a purple hue near the center of the colony. Under microscopy, abundant hyaline, canoe-shaped macroconidia with four to seven septations (55.11 ± 6.20 × 10.21 ± 0.61 µm) were observed consistent with morphology described for F. oxysporum (McPartland and Hillig 2004). Inoculations with F. oxysporum had no wounding, and wilt symptoms were observed on inoculated plants within 2 weeks of inoculation, vascular discoloration was observed, and F. oxysporum was reisolated from all inoculated plants. BLASTn searches based on EF1-α, TUB2, and RPB2 all agreed that the representative pure isolate was most similar to F. oxysporum (Table 1), and this was supported by the multilocus phylogenetic tree with 100% bootstrap support (Fig. 1C).

    Fusarium foliar and flower blight.

    Fusarium foliar and flower blight was observed on 16 and 20% of samples submitted to the PDIC in 2017 and 2018, respectively. Symptoms were observed beginning in August of each year and during flower drying. Abundant hyphae were observed on the surface of floral parts (Fig. 2F and G), and the tips of flower parts had light brown necrotic lesions. Leaf symptoms included circular, light-brown lesions (approximately 1-cm diameter) with a dark brown margin (Fig. 2H). As leaf lesions age, the center of the lesion becomes gray-brown. Within the center of lesions, abundant, hyaline, canoe-shaped macroconidia with three to seven septations were observed (F. equiseti: 21.22 ± 5.75 × 6.81 ± 0.81 µm; F. graminearum: 29.31 ± 6.20 × 3.86 ± 1.25 µm). In culture, both isolates produced abundant white hyphae and macroconidia, and media became fuchsia pink as the isolates expanded. Morphology of isolates from field samples and cultural characteristics were consistent with Fusarium spp. (Carmichael et al. 1980; Seifert and Gams 2011). Two representative isolates were selected for sequencing due to variability of fungal growth and spore size among the isolates collected. Lesions were observed when plants were inoculated with both F. equiseti and F. graminearum with no physical wounding. For the putative F. equiseti isolate, BLASTn searches using EF1-α, TUB2, and RPB2 agreed that it was most similar to F. equiseti. However, for the putative F. graminearum isolate, queries based on TUB2 and RPB2 agreed on similarity to F. graminearum, whereas queries based on EF1-α indicated greatest similarity to F. equiseti (Table 1). The multilocus phylogeny based on all three loci indicated that the putative F. equiseti isolate grouped with F. equiseti with 98% bootstrap support, and the putative F. graminearum isolate grouped most closely with F. graminearum with 97% bootstrap support (Fig. 1C).

    Fusarium stem canker.

    Fusarium stem canker (F. graminearum) was observed on plants (8%) from field production in early summer (July 2018) (Table 1). Plants were wilted and collapsed at the lower stem (Fig. 2I). A cross-section of stem tissues revealed brown to pink-brown discoloration of vascular tissues on either side of the girdled stem. Macroconidia and microconidia were observed within discolored tissues of the stem. In culture (PDA), abundant white to eggshell colored hyphae were produced abundantly, and media became fuchsia pink colored as the colony expanded. Morphology was consistent with F. graminearum as described above (Carmichael et al. 1980; Seifert and Gams 2011). BLASTn searches for EF1-α, TUB2, and RPB2 agreed that the representative isolate was most similar to F. graminearum (Table 1), and this was supported by the multilocus phylogeny with 97% (Fig. 1C).

    Helminthosporium leaf spot.

    Helminthosporium leaf spot (E. rostratum) was observed on samples sent to PDIC in 2017 (16%) and 2018 (11%). Symptoms were observed season-long after transplant. Lesions were found on leaves and stems, which were round, brown to black, with darkened margins. Within each lesion, abundant conidia (75.64 ± 8.31 × 15.61 ± 1.41 µm) were observed (Thiessen and Schappe 2019). BLASTn queries for ITS1 and RPB2 both found greatest similarity to E. rostratum (Table 1). The multilocus phylogeny was in agreement, showing that the representative isolate grouped most closely with Setosphaeria rostrata, a synonym, with 75% bootstrap support (Fig. 1B).

    Southern blight.

    Southern blight (Sclerotium rolfsii) was found on five samples submitted to the PDIC in 2017 (5%) and 2018 (4%). Symptoms were observed midseason, when conditions in this growing region become hotter. Symptoms of whole-plant wilting were observed on reproductive plants (Fig. 2J). Closer inspection of roots and lower stems showed abundant hyphae (Fig. 2K), and small, round, russet-brown sclerotia were observed. No spores were present when found on plant tissues or in culture. Morphology of hyphae and damages were consistent with previous reports (McPartland et al. 2000). BLASTn searches based on ITS1/ITS2 and 18S rRNA agreed that the representative isolate was most similar to Athelia rolfsii, the anamorph, and the multilocus phylogeny agreed with 100% bootstrap support (Fig. 1E).

    Rhizoctonia root rot and sore shin.

    Rhizoctonia infections were identified on root and crowns of 3% of samples submitted to the PDIC in 2018 during the month of July. Symptoms included wilting of plants, and necrotic lesions at the base of the plants just above the soil line. Under microscopy, hyphae obtained from necrotic lesions were pigmented and septate, and they branched at 90° angles with a basal constriction from adjacent septa. In culture, colonies were cream-white colored, and irregularly shaped, medium to dark brown sclerotia were formed. BLASTn queries using ITS and 18S rRNA agreed that the representative isolate was most similar to Rhizoctonia solani (Table 1).

    Root-knot nematode.

    Root-knot nematode (Meloidogyne sp.) was identified in one sample submitted to the PDIC (three total affected plants submitted) in August 2018, and this is the only field-identified nematode injury on hemp to date. Plants were minimally infested but had some associated stunting. Roots had characteristic galls containing females inside of the root tissues. Egg masses were observed on the surface of females, and eggs with developing juveniles were observed upon maceration of egg masses under the microscope. PCR assays indicated that females assessed were all Meloidogyne incognita, and mixed populations were not found present within this field.

    Abiotic Disorders from Survey Tissues

    PDIC abiotic disorders.

    Abiotic samples increased in the PDIC from four in 2017 to 30 in 2018, for a total of 34 samples. Of the abiotic disorders reported, root binding and excess water were the most common disorders observed (31 of 107 samples submitted to the PDIC) (Fig. 3).

    FIGURE 3

    FIGURE 3 Abiotic disorders were characterized on industrial hemp with symptoms including interveinal yellowing and necrosis caused by manganese toxicity (A and B), necrosis and crisping of leaf edges caused by boron toxicity (C, photo credit: Jeremy Barnes), root binding (D), leaf-edge burning and yellowing of oldest tissues caused by potassium deficiency (E), and chlorosis of new foliar tissues from clomazone injury (F).

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    Excess water.

    Two hurricanes impacted the Southeast United States in 2018, Hurricane Florence and Hurricane Michael. Anticipated hurricane impacts forced many growers to harvest their industrial hemp crop early (n = 34; 13.9%), impacting their yield. Those that did not harvest ahead of the storms suffered significant losses due to wind damage (n = 69; 28.2%) and acute flooding or chronic water-logging (n = 49; 20%). Much of North Carolina experienced double the normal annual rainfall in 2018 (data not shown), and 39.2% of producers reported weather as their number one production problem in 2018 (n = 96).

    Nutrient disorders.

    Of the 573 samples assessed by the ASD, 85% exhibited symptoms of nutrient disorders (Figs. 4 and 5). Of the samples tested, 41.78% were within recommendation thresholds of N, 32.52% of P, 18.88% of K, 40.38% of Ca, 37.76% of Mg, 60.66% of S, 29.90 of Fe, 14.69% of Mn, 21.85% of Zn, 29.51% of Cu, and 33.39% of B. Nutrient excesses were documented in samples submitted to the ASD including Ca, Mg, S, Fe, Mn, Cu, and B (Table 2). Additional samples with suspected nutrient disorders were reported to and documented by state extensions specialists, the most common ones being boron and manganese toxicity and potassium deficiencies (Fig. 3). Manganese toxicity is characterized by interveinal yellowing followed by interveinal necrosis (Fig. 3A and B). Boron toxicity is characterized by necrosis and crisping of leaf edges (Fig. 3C). Veins may remain dark green in mild cases. Potassium deficiency was also noted from several field samples and images submitted by growers. Potassium deficiency is characterized in industrial hemp by burning on the edges of the leaves and yellowing in a speckling pattern on the oldest leaf tissues. In severe cases, the entire leaf is yellow and the edges are necrotic (Fig. 3E).

    FIGURE 4

    FIGURE 4 Nutrient content (percent per gram of plant tissue) found for independent samples (x axis) submitted to the North Carolina Department of Agriculture and Consumer Services Agronomic Services Division (n = 572), with many plants in excess of recommended nutrient content (values above solid line) and insufficient recommended nutrient content (values below dashed line) for N, P, K, S, Ca, and Mg.

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    FIGURE 5

    FIGURE 5 Nutrient content (parts per million per gram of plant tissue) found for independent samples (x axis) submitted to the North Carolina Department of Agriculture and Consumer Services Agronomic Services Division (n = 572), with many plants in excess of recommended nutrient content (values above solid line) and insufficient recommended nutrient content (values below dashed line) for Fe, Mn, Zn, Cu, and B.

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    TABLE 2 Percent of samples (n = 572) in 2017 and 2018 assayed by the North Carolina Department of Agriculture Agronomic Services Division that fall within and out of threshold recommendations for Cannabis sativa set by the Plant Analysis Handbook (Bryson et al. 2014)

    Root binding.

    In 2018, 40% of growers (n = 100) self-reported root binding of transplants purchased in North Carolina. Once transplanted, industrial hemp did not produce additional adventitious roots to overcome root binding in the field (Fig. 3D). An additional problem observed with root binding was secondary infections from Fusarium spp., Pythium spp., and other root-rotting pathogens (clinic sample observation).

    Herbicide injury.

    Herbicide injury was assessed on four samples in 2017 and 2018. Injury from the carotenoid biosynthesis-inhibiting herbicide clomazone was observed in a commercial field, causing bleaching of newly formed tissues following exposure (Fig. 3F). Clomazone is a soil-residual carotenoid biosynthesis inhibitor and can carry over from previous crops or on land meant for another crop that is abandoned and replanted to hemp (Senseman 2007). In this case, the land was reserved for tobacco planting and treated with clomazone ahead of tobacco. It was then planted to hemp instead, causing severe injury from which the hemp did not recover. Many other herbicides have the potential to injure industrial hemp through carryover and drift, including paraquat; however, only clomazone symptomology was documented in the years of this survey.

    Survey of Hemp Producers

    Overall, 49.6% reported diseases as one of their pest pressures; however, only 26.6% of producers reported utilizing the PDIC to submit samples for professional identification. Table 3 represents the diseases self-reported by industrial hemp producers through the annual survey. Other production issues identified included weeds, insects (particularly caterpillars and russet mites), deer, birds, and abiotic disorders. Root binding was reported by 41.3% of producers, and 4.8% reported it as their number one problem. Other abiotic disorders reported were nutrient deficiencies (26.6%), excess water (32.54%), drought stress (13.9%), and pH issues (0.8%). Diseases were reported by 11.4% of producers as the number one problem in 2018 (n = 28).

    TABLE 3 Diseases as self-reported by industrial hemp (Cannabis sativa L.) producers from the 2018 producer survey conducted by the North Carolina Department of Agriculture Industrial Hemp Commission

    Potential Impacts of Survey Results

    Economical industrial hemp production may be threatened by significant disease and abiotic pressures. This research confirmed 13 distinct disease-causing agents, two environmentally caused abiotic disorders, and three nutritional disorders in the two years of research (Tables 1 and 2). Many diseases and abiotic disorders have been previously documented in North America (McPartland 1983, 1994, 1995a, 1995b, 1995c, 1995d; McPartland and Hillig 2004; McPartland and Hughes 1994; Pépin et al. 2018; Punja et al. 2018); however, this research identified several new hemp disease-causing microorganism species that had not yet been documented in previous studies (Table 1, Fig. 1). The potential for these limiting factors to continue to cause widespread losses may require changes to production practices depending upon regional climate conditions. For example, communications with other similar growing regions report different Helminthosporium fungi being predominant (e.g., Bipolaris spp. or Drechslera spp.) in similar hemp production systems (N. Gauthier and C. Johnson, personal communications). Although cultural disease control practices may be similar for all leaf spot pathogens, fungicide selection may differ based on efficacy in controlling local fungal species.

    Current management practices vary by end-product use for industrial hemp. In this region, therapeutic hemp varieties are being cultivated most widely in raised bed systems. Production on plasticulture may inadvertently favor soilborne diseases, including Fusarium wilt and root rots or white mold (McPartland and Hillig 2004; McPartland et al. 2000). This may be due to continued saturated soil conditions, protection of soil inoculum from desiccation, and stress conditions on the plant (McPartland and Hillig 2004; McPartland et al. 2000). Conversely, in dry environments, production in plasticulture with drip irrigation systems may facilitate water management practices that favor plant growth and development without increasing foliar diseases such as bacterial leaf spot or Fusarium foliar and flower blights (McPartland et al. 2000). In grain and fiber production with dense, direct-seeded hemp plants, damping off pathogens may be favored in cool, wet environments, favoring Pythium spp., Thielaviopsis spp., and Fusarium spp., or warm, dry environments, favoring Rhizoctonia spp. Continued evaluation of the circumstances that induce or mitigate disease-favoring conditions or plant-stress conditions is needed to anticipate the economic impact that many of these diseases will have on the production of industrial hemp.

    Environmental conditions that promote plant stress directly impact plant health (Doorenbos and Kassam 1979; Lafever 1981) and indirectly impact disease susceptibility of plants (Graham 1983; Schoeneweiss 1975). Unlike other transplanted crops in the region, hemp plants do not generate new roots above roots developed in the greenhouse prior to setting. In 2018, many hemp transplants that had roots bound up in transplant-cell volumes were transplanted later than anticipated due to poor weather conditions, which is similar to other ornamental crops (De Lojo et al. 2017; Di Benedetto 2011; van Iersel 1997). These conditions may have increased the development of Fusarium wilt, southern blight, and Rhizoctonia root and crown rot of reproductive plants in 2018 (Table 1), which could be a continuing issue in years with delayed planting (Emmett et al. 2014). Additionally, significant rainfall conditions in 2018 also impacted the growth and development of industrial hemp (L. Thiessen, personal observation). The damage associated with saturated soil conditions limited root development and caused roots to lose epidermal tissues. Hemp may not be adapted to conditions with heavy saturation, unlike other tropical plants produced in the southeast (e.g., tobacco). Although the origins of industrial hemp development are still open to speculation, Central Asia has been cited (Russo 2007). The climate in Central Asia has less consistent precipitation and a drier climate than the southeast United States (Russo 2007). Saturated roots that were evaluated in the PDIC often had secondary infections by bacteria or Fusarium spp. (16%). These diseases may continue to be associated with plants under water stresses. The results from this study are primarily diseases of transplanted, therapeutic hemp cultivars, and grain or fiber hemp types may respond differently to conditions described in this work. Additionally, these hemp types are direct seeded, as opposed to transplanted, in high-density plantings, which may influence disease susceptibility. An additional complication to saturated conditions observed may be the impact of unknown nutritional requirements of industrial hemp and the influence of environmental conditions on the uptake of nutrients under poor conditions.

    Several nutrient deficiencies and toxicities (>58% of samples assessed) were observed in industrial hemp in both years of the pilot program. Nutrient availability may have been significantly impacted by extreme weather conditions that brought abundant rainfall to the region in 2018. Furthermore, stresses attributed to nutrient deficiencies and toxicities may influence hemp cannabinoid and terpene profiles (Haney and Kutscheid 1973; Latta and Eaton 1975), influencing marketability. Several volatile compounds produced by hemp have also demonstrated antifungal activity (Wanas et al. 2016), and nutrient stress may influence the availability of these compounds when plants are challenged by plant pathogens. An important nutrient management limitation for producers is the lack of up-to-date nutrient recommendations for industrial hemp. Currently, the NCDA&CS ASD utilizes recommendations from the Plant Analysis Handbook (Bryson et al. 2014); however, these recommendations derive from survey leaf ranges measured in greenhouse nursery production for which neither variety, number, nor crop type (grain, fiber, CBD, or THC) are reported. Preliminary observations by NCDA&CS suggest that these ranges may not accurately describe acceptable nutrient levels in leaf tissue under other production systems and between varieties. Further evaluation of nutrient requirements is needed to develop adequate nutritional requirements for industrial hemp production.

    Although the results from this trial provide insight into potential diseases and disorders of industrial hemp, other diseases and disorders may cause significant losses for producers in dissimilar climatic regions. For example, Cercospora cf. flagellaris was found on industrial hemp in Kentucky (Doyle et al. 2019), but Cercospora spp. have not yet been economically impactful in the coastal, southeast region of hemp production. Additionally, although Bipolaris spp. have been identified in other regions (Szarka et al. 2020), this pathogen has not yet been confirmed in North Carolina, where E. rostratum appears to have greater impact (Thiessen and Schappe 2019). In addition to regional variability of pressures, the biology of new and emerging pathogens is poorly understood. Mitigation of diseases and disorders may have broad impacts on the industrial hemp trade and human health. Similar crops that are smoked (e.g., tobacco), oils extracted (e.g., essential oil crops), or consumed (e.g., wheat and barley) are subject to restrictions on both pesticides and the presence of harmful mycotoxins. Given the similarities in uses among these crops, restrictions on the presence of both pesticide residues and mycotoxins will likely be imposed in the marketplace. Managing these diseases and disorders will require extensive testing in industrial hemp to evaluate the efficacy and impacts of mitigation strategies.

    Acknowledgments

    We thank the technical support of Y. I. Rosado Rivera and A. L. Joyce.

    The author(s) declare no conflict of interest.

    Literature Cited

    The author(s) declare no conflict of interest.

    Funding: The authors thank the North Carolina Department of Agriculture, North Carolina State University, and USDA National Institute of Food and Agriculture Project No. NC02617 for financial support of this research.