Characteristics of the Diploid, Triploid, and Tetraploid Versions of a Cannabigerol-Dominant F1 Hybrid Industrial Hemp Cultivar, Cannabis sativa 'Stem Cell CBG'.

Hemp (Cannabis sativa L.) has recently become an important crop due to the growing market demands for products containing cannabinoids. Unintended cross-pollination of C. sativa crops is one of the most important threats to cannabinoid production and has been shown to reduce cannabinoid yield. Ploidy manipulation has been used in other crops to improve agronomic traits and reduce fertility; however, little is known about the performance of C. sativa polyploids. In this study, colchicine was applied to two proprietary, inbred diploid C. sativa inbred lines, 'TS1-3' and 'P163', to produce the tetraploids 'TS1-3 (4x)' and 'P163 (4x)'. The diploid, triploid, and tetraploid F1 hybrids from 'TS1-3' × 'P163', 'TS1-3 (4x)' × 'P163', and 'TS1-3 (4x)' × 'P163 (4x)' were produced to test their fertilities, crossing compatibilities, and yields. The results indicated a reduction in fertility in the triploids and the tetraploids, relative to their diploid counterparts. When triploids were used as females, seed yields were less than 2% compared to when diploids were used as females; thus, triploids were determined to be female infertile. The triploids resulting from the crosses made herein displayed increases in biomass and inflorescence weight compared to the diploids created from the same parents in a field setting. Statistical increases in cannabinoid concentrations were not observed. Lastly, asymmetric crossing compatibility was observed between the diploids and the tetraploids of the genotypes tested. The results demonstrate the potential benefits of triploid C. sativa cultivars in commercial agriculture.

First report of Sclerotinia sclerotiorum causing stem canker on Cannabis sativa L. in Oregon.

In August of 2020, plants of Cannabis sativa L. grown in hoop houses at two farms located in Benton County, Oregon exhibited wilting and chlorosis, followed by shoot necrosis. Symptomatic plants had dry, tan-brown lesions or cankers, often accompanied by large, round to irregular or ribbon-shaped, black sclerotia and/or profuse white mycelial growth. Lesions or cankers were observed on the stems at both the plant crown (soil) level and higher in the canopy; flower infections were not observed. Sclerotia were removed from two infected plants and placed on potato dextrose agar (PDA) at room temperature. Fast-growing, pure white, largely appressed, sterile mycelium grew radially from plated sclerotia. Hyphal tips were transferred to obtain a pure culture. Additional sclerotia, solitary and aggregate, approximately 30 to more than 50 per plate, exhibiting identical features to those observed on plant tissue, formed in culture 6-7 days following transfer and ranged in size from 2 to 11 mm in length or width (n=50). Mycelia were aseptically harvested from cultures for DNA extraction (Quick-DNA Plant/Seed Miniprep Kit, Zymo Research). Primers ITS1-F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) were used to amplify the internal transcribed spacer region (ITS) and primers G3PDHfor and G3PDHrev were used to amplify the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene (Staats et al. 2005) from a single isolate, LAS01. The ITS region from LAS01 (MW079844) shared 100 to >99% homology to several Sclerotinia species isolates in GenBank. The LAS01 G3PDH gene (MW082601), shared >99% and 100% homology with S. sclerotiorum type specimens strains 484 (GenBank accession no. AJ705044) and 1980 (JQ036048), respectively, and only 97% and 96% sequence identity with S. minor (KF878364) and S. trifoliorum (KF878375), respectively. A phylogenetic tree (presented as an eXtra) identifies LAS01 as S. sclerotiorum. To confirm pathogenicity, isolate LAS01 was grown on PDA at room temperature. After 48 hours, 4mm plugs were cut from the colony and placed mycelium-side down onto the main stems of five healthy C. sativa plants that had been grown for approximately six weeks from rooted cuttings and secured using a minutien pin. Uncolonized PDA plugs placed on the stem of the same plants several leaf nodes away were used as controls. Plants were incubated at room temperature in a grow tent under 24-hour light and 70-95% humidity conditions. Elongate, tan-brown lesions were observed at the inoculation sites 4-5 days post inoculation; stems at mock inoculated sites remained green. After six days, tissue was excised from the margin of each lesion, surface sterilized with 1% NaOCl, rinsed in sterile water, and placed onto PDA. Resultant fungal growth was confirmed to be S. sclerotiorum based on morphology. Isolation attempts were also made from mock inoculations; no fungal growth was observed. Trials were repeated on two additional cultivars with similar results. This report is the first of S. sclerotiorum on C. sativa in Oregon; the only peer-reviewed reports that could be located for S. sclerotiorum on C. sativa in the United States were from host indices in Montana (Anon. 1960; Shaw 1973) and references cited by McPartland (1996). Sclerotinia sclerotiorum has been reported in Canada on hemp-type C. sativa (Bains et al. 2000). The economic impact of S. sclerotiorum on the emerging C. sativa industry in Oregon and the United States remains unclear.

First report of Powdery Mildew Caused by Golovinomyces ambrosiae on Cannabis sativa in Oregon.

Oregon is the second largest producer of hemp in the United States with 25,900 ha of hemp licensed to growers in 2019, a nearly six-fold increase over the previous year (Perkowski 2019, Capital Press). Industrial hemp has a wide range of uses including textiles to nutritional supplements; in Oregon, hemp has become one of the most economically promising crops and is mainly cultivated for cannabidiol (CBD) production. Between 2018 and 2019, multiple independent greenhouse growers in western Oregon reported powdery mildew-like signs and symptoms on leaves and buds of several Cannabis sativa cultivars, including 'Cherry Wine'. Signs of the disease started as small, white, powdery patches, typically on the adaxial sides of leaves, and progressed to coalescent colonies on leaves, stems, and buds. Fungi present on diseased tissues had unbranched hyaline conidiophores that measured 140 to 250 µm and grew erect from caulicolous and amphigenous mycelium (n = 15). Foot cells were cylindrical, often tapered at one or both ends, and measured 80 to 117 × 9.5 to 11.9 µm (n = 15). Conidia were catenescent, hyaline, ellipsoidal to barrel-shaped, lacked fibrosin bodies, and measured 24 to 34 × 12 to 18 µm (n = 50). No chasmothecia were observed. Morphological observations overlapped with several Golovinomyces spp. Including G. ambrosiae, G. cichoracearum, and G. spadiceus (Braun and Cook 2012). Identification was confirmed by bidirectional sequencing and phylogenetic analysis of 1,457 nucleotides from the concatenated internal transcribed spacer (ITS), 28S large ribosomal subunit, and beta-tubulin (TUB2) regions of two isolates using primer pairs ITS1/ITS4 and NL1/LR5, and TubF1/TubR1 respectively (Mori et al. 2000, Qiu et al. 2020, Vilgalys and Hester 1990, White et al. 1990; GenBank Acc. No.: MW248121 to MW248124, MW265971 to MW265972). The Oregon hemp isolates grouped (bootstrap value = 100) in a monophyletic clade with G. ambrosiae accessions from Qiu et al. (2020). Pathogenicity was confirmed by transferring conidia by leaf rub inoculation onto 2-to 4-week-old 'Cherry Wine' potted plants and incubated outdoors at 12 to 22°C. Control plants were mock-inoculated using healthy leaves. Powdery mildew symptoms developed on inoculated plants approximately 14 to 21 days later; control plants were asymptomatic. Identification was confirmed by morphological characterization and sequencing using the aforementioned primers. The hemp isolates were also able to infect detached leaves of Humulus lupulus 'Symphony' via similar inoculations; however, colony development on 'Symphony' was slow and sporulation sparse as was reported by Weldon et al. (2020). Golovinomyces spp. have also been reported on hemp in Kentucky (Szarka et al. 2019), Ohio (Farinas and Peduto Hand 2020), and New York (Weldon et al. 2020). Although reported as G. spadiceus, these reports are also likely G. ambrosiae according to new taxonomic revision of the genus (Qiu et al. 2020). This is the first known report of Golovinomyces ambrosiae causing powdery mildew on hemp in Oregon (OSC 171893). While powdery mildew on hemp currently appears most severe in protected cultivation, rapid expansion of hemp cultivation and introduction of new CBD varieties throughout Oregon could lead to increased powdery mildew risk in outdoor cultivation.

SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in Cannabis sativa L.

The regulation of cannabinoid synthesis in Cannabis sativa is of increasing research interest as restrictions around the globe loosen to allow the plant's legal cultivation. Of the major cannabinoids, the regulation of cannabigerolic acid (CBGA) production is the least understood. The purpose of this study was to elucidate the inheritance of CBGA dominance in C. sativa and describe a marker related to this chemotype. We produced two crossing populations, one between a CBGA dominant cultivar and a tetrahydrocannabinolic acid (THCA) dominant cultivar, and one between a CBGA dominant cultivar and a cannabidiolic acid (CBDA) cultivar. Chemical and genotyping analyses confirmed that CBGA dominance is inherited as a single recessive gene, potentially governed by a non-functioning allelic variant of the THCA synthase. The "null" THCAS synthase contains a single nucleotide polymorphism (SNP) that may render the synthase unable to convert CBGA to THCA leading to the accumulation of CBGA. This SNP can be reliably used as a molecular marker for CBGA dominance in the selection and breeding of C. sativa.

The History of Botrytis Taxonomy, the Rise of Phylogenetics, and Implications for Species Recognition.

Botrytis is one of the oldest, most well studied, and most economically important fungal taxa. Nonetheless, many species in this genus have remained obscured for nearly 300 years because of the difficulty in distinguishing these species by conventional mycological methods. Aided by the use of phylogenetic tools, the genus is currently undergoing a taxonomic revolution. The number of putative species in the genus has nearly doubled over the last 10 years and more species are likely to be discovered in the future. The implementation of phylogenetic species recognition concepts in Botrytis is providing for more resolution on the relatedness among species than ever before, and this has helped to overcome issues in historical species recognition using morphology, sexual crosses, and pathogenicity tests. Meanwhile, the use of genetic tools is helping to reveal surprising insight into this archetypal necrotroph's behavior, making these approaches increasingly important in species recognition and identification. As Botrytis taxonomy continues to evolve at a rapid pace, researchers should be encouraged to continue to employ the powerful tool of phylogenetics while considering how it fits into a larger framework of classical Botrytis species recognition. Starting points for discussion on how to move forward with Botrytis species recognition are included herein, with an emphasis on the implications and utility of new species descriptions.

Genetic analysis reveals unprecedented diversity of a globally-important plant pathogenic genus.

Genus Botrytis contains approximately 35 species, many of which are economically-important and globally-distributed plant pathogens which collectively infect over 1,400 plant species. Recent efforts to genetically characterize genus Botrytis have revealed new species on diverse host crops around the world. In this study, surveys and subsequent genetic analysis of the glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and necrosis and ethylene-inducing proteins 1 and 2 (NEP1 and NEP2) genes indicated that Botrytis isolates collected from peony fields in the United States contained more species diversity than ever before reported on a single host, including up to 10 potentially novel species. Together, up to 16 different phylogenetic species were found in association with peonies in the Pacific Northwest, which is over a third of the total number of species that are currently named. Furthermore, species were found on peonies in Alaska that have been described on other host plants in different parts of the world, indicating a wider geographic and host distribution than previously thought. Lastly, some isolates found on peony share sequence similarity with unnamed species found living as endophytes in weedy hosts, suggesting that the isolates found on peony have flexible lifestyles as recently discovered in the genus. Selected pathogenicity, growth, and morphological characteristics of the putatively new Botrytis species were also assessed to provide a basis for future formal description of the isolates as new species.

Botrytis euroamericana, a new species from peony and grape in North America and Europe.

A novel species of Botrytis isolated from peony in Alaska, USA, and grape in Trento District, Italy, was identified based on morphology, pathogenicity, and sequence data. The grape and peony isolates share sequence homology in the glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and necrosis- and ethylene-inducing protein 1 and 2 (NEP1 and NEP2) genes that place them in a distinct group closely related to B. aclada, a globally distributed pathogen of onions. Genetic results were corroborated with morphological and pathogenicity trials that included two isolates of B. cinerea and two isolates of B. paeoniae from peony in Alaska and one isolate of B. aclada. The authors observed differences in colony and conidia morphology and ability to cause lesions on different host tissues that suggest that the grape and peony isolates represent a distinct species. Most notably, the grape and peony isolates did not colonize onion bulbs, whereas B. aclada readily produced lesions and prolific sporulation on onion tissue. The new species Botrytis euroamericana is described herein.