Journal:Pathogens and molds affecting production and quality of Cannabis sativa L.

Full article title	Pathogens and molds affecting production and quality of Cannabis sativa L.
Journal	Frontiers in Plant Science
Author(s)	Punja, Zamir K.; Collyer, Danielle; Scott, Cameron; Lung, Samantha; Holmes, Janesse; Sutton, Darren
Author affiliation(s)	Simon Fraser University
Primary contact	Email: punja at sfu dot ca
Editors	Smith, Donald L.
Year published	2019
Volume and issue	10
Article #	1120
DOI	10.3389/fpls.2019.01120
ISSN	1664-462X
Distribution license	Creative Commons Attribution 4.0 International
Website	https://www.frontiersin.org/articles/10.3389/fpls.2019.01120/full
Download	https://www.frontiersin.org/articles/10.3389/fpls.2019.01120/pdf (PDF)

Abstract

Plant pathogens infecting marijuana (Cannabis sativa L.) plants reduce growth of the crop by affecting the roots, crown, and foliage. In addition, fungi (molds) that colonize the inflorescences (buds) during development or after harvest, and which colonize internal tissues as endophytes, can reduce product quality. The pathogens and molds that affect C. sativa grown hydroponically indoors (in environmentally controlled growth rooms and greenhouses) and field-grown plants were studied over multiple years of sampling. A polymerase chain reaction (PCR) assay using primers for the internal transcribed spacer region (ITS) of ribosomal DNA confirmed identity of the cultures. Root-infecting pathogens included those from the Fusarium genus (Fusarium oxysporum, Fusarium solani, and Fusarium brachygibbosum) and the Pythium genus (Pythium dissotocum, Pythium myriotylum, and Pythium aphanidermatum), which caused root browning, discoloration of the crown and pith tissues, stunting and yellowing of plants, and in some instances, plant death. On the foliage, powdery mildew, caused by Golovinomyces cichoracearum, was the major pathogen observed. On inflorescences, Penicillium bud rot (caused by Penicillium olsonii and Penicillium copticola), Botrytis bud rot (Botrytis cinerea), and Fusarium bud rot (F. solani, F. oxysporum) were present to varying extents. Endophytic fungi present in crown, stem, and petiole tissues included soil-colonizing and cellulolytic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium, and Fusarium. Analysis of air samples in indoor growing environments revealed that species of Penicillium, Cladosporium, Aspergillus, Fusarium, Beauveria, and Trichoderma were present. The latter two species were the result of the application of biocontrol products for control of insects and diseases, respectively. Fungal communities present in unpasteurized coconut (coco) fiber growing medium are potential sources of mold contamination on Cannabis plants. Swabs taken from greenhouse-grown and indoor buds pre- and post-harvest revealed the presence of Cladosporium and up to five species of Penicillium, as well as low levels of Alternaria species. Mechanical trimming of buds caused an increase in the frequency of Penicillium species, presumably by providing entry points through wounds or spreading endophytes from pith tissues. Aerial distribution of pathogen inoculum and mold spores and dissemination through vegetative propagation are important methods of spread, and entry through wound sites on roots, stems, and bud tissues facilitates pathogen establishment on Cannabis plants.

Keywords: diseases, plant pathogens, epidemiology, post-harvest molds, fungi, root infection, endophytes

Introduction

Cannabis sativa L., a member of the family Cannabaceae, is cultivated worldwide as hemp (for fiber, seed, and oil) and marijuana (referred to hereon as cannabis) for medicinal and psychotropic effects. The pathogens affecting production of hemp have been described and include fungal, bacterial, viral, and nematode species. In contrast, the pathogens affecting cannabis have not been extensively studied, and the different growing environments, cultivation methods, as well as differences among the strains or genetic selections of hemp and cannabis can influence disease development. This requires that studies on the pathogens potentially affecting Cannabis plants be conducted so that methods to manage emerging diseases and molds can be developed. Cannabis plants are propagated from cuttings that are rooted and grown vegetatively, following which they are transferred to conditions of specific reduced lighting regimes (photoperiod) to induce flowering. Flower buds are harvested, dried, and stored in vacuum-sealed bags or sealed plastic or glass containers prior to distribution. Fungal infection of roots can occur at any time during the production cycle, while colonization of flower buds generally occurs during the later stages of flower development and can be manifested as a pre-harvest or post-harvest bud rot. In addition, foliar pathogens may infect the plant at any stage during its production.

The objectives of this research were to determine the prevalence of root-infecting, foliar-infecting, and flower-infecting fungi affecting Cannabis plants grown in indoor environments, in greenhouses, and under field conditions to obtain a better understanding of the diseases affecting this plant. In addition, the incidence of molds in the growing environments, and on pre-harvest and post-harvest inflorescences, was assessed. Cultural methods for isolation, and morphological and molecular methods for identification, were used in this study. More than 22 different fungal and oomycete species and their associated effects on Cannabis plants grown indoors and outdoors are presented.

Materials and methods

Isolation of pathogens and molds from cannabis tissue

A range of tissue samples were obtained from Cannabis plants grown in indoor controlled environments (two locations) and greenhouse-grown plants (one location) of various Cannabis strains (e.g., Moby Dick, Hash Plant, Pink Kush, Pennywise, Girl Scout Cookies) under licensed commercial production, as well as from field-grown plants (one location) (Figure 1). They included roots, crown tissues, leaves, and flower buds. Samples either displayed symptoms of browning and were presumed to be infected by pathogens or were symptomless. Tissues were sampled at various times during growth of the plants, ranging from early stages of propagation (1–3 weeks old) (Figures 1A, B), to advanced vegetative growth (3–6 weeks of age) (Figures 1C, D), and then to plants that were in full flower (7–14 weeks of age) (Figures 1E, F). Samples were also obtained of harvested buds before and after they were dried, from indoor and field productions. These tissue samples were obtained over a duration of three years, from 2016 to 2018. They were taken at multiple times during the production cycle, and at varying time periods, depending on the pathogen of interest. Each sampling time had a minimum of five replicate samples. All plants were grown indoors and in greenhouses using either Rockwool blocks as a substrate or in coco fiber (coco coir) derived from different commercial suppliers. Plants were watered through an automated irrigation system with individual emitters for each plant. They were provided with the appropriate nutrient regimes and lighting conditions as required for commercial production.

Fig. 1 Production systems used for Cannabis sativa plants that were sampled in this study. (A) Rooting of vegetative cuttings in rockwool plugs containing peat in the central plugs. Cuttings are left for two weeks under supplemental lighting to initiate rooting. (B) Growth of plants in a hydroponic production system with clay pellets as a substrate. Plants are in the early stages of vegetative development. (C) Six-week-old plants in a hydroponic production system ready for transfer from vegetative growth to induction of flowering through controlled photoperiod and light intensity regimes. (D) Greenhouse hydroponic production system using coco fiber blocks as a substrate, showing a plant in the early stages of flower development. (E, F) Field production of C. sativa in raised fabric pots under outdoor conditions. (E) Plants in early stages of flower development. (F) Close-up of shoots bearing flowers. Figure 1E reproduced from Punja et al. 2018.

A total of around 220 plants were sampled in the study. Among these, around 90 originated from the two indoor production facilities and 120 from the greenhouse facility, all located in British Columbia. In 2019, an additional five samples of diseased tissues were received from one production facility in Ontario, showing symptoms of root browning and stem discoloration, and five samples of bud tissues originated from a field production site in British Columbia in 2018. Plants with visible symptoms of disease were photographed.

Small tissue pieces of approximately 0.5 cm in length for roots or 0.2–0.4 cm² for leaves or flower buds were surface-disinfected by dipping them in a 0.5% NaOCl solution for 30 seconds, followed by 20 seconds in 70% EtOH, rinsed thrice in sterile water, blotted on sterile paper towels, and plated onto Potato Dextrose Agar (PDA, Sigma Chemicals, St. Louis, MO) amended with 100 mg/L of streptomycin sulfate (PDA+S). Dishes containing the tissues were incubated under ambient laboratory conditions (temperature range of 21–24°C with 10–14-hours/day fluorescent lighting) for 5–10 days. Emerging colonies were recorded and transferred to fresh PDA+S dishes for subsequent identification to the genus level using morphological criteria, including colony color and size, as well as microscopic examination of spores. Species-level identification was done by polymerase chain reaction (PCR) using the primers ITS1F-ITS4 (ITS1-F 5’-CTTGGTCATTTAGAGGAAGTAA-3’ and ITS4 5’-TCCTCCGCTTATTGATATGC-3’). The resulting sequences were compared to the corresponding ITS1-5.8S-ITS2 sequences from the National Center for Biotechnology Information (NCBI) GenBank database to confirm species identity using only sequence identity values above 99%. These sequences have been deposited in GenBank. Pathogenicity tests were conducted for representative isolates (a minimum of two) of Fusarium oxysporum and Fusarium solani recovered from roots, as well as two isolates each of Botrytis cinerea and Penicillium olsonii recovered from flower buds, following the methods described by Punja and Rodriguez and Punja.

Scanning electron microscopy

Powdery mildew infection of leaves, and infection of buds by P. olsonii and F. oxysporum following artificial inoculation with spores, as well as stem segments showing pith tissues, was prepared for scanning electron microscopy (SEM) as follows. Tissue segments of approximately 0.5 cm² were adhered to a stub using a graphite-water colloidal mixture (G303 Colloidal Graphite, Agar Scientific, UK) and Tissue-Tek (O.C.T. Compound, Sakura Finetek, NL). The sample was submerged in a nitrogen slush for 10–20 seconds to rapidly freeze it. After freezing, the sample was placed in the preparation chamber of a Quorum PP3010T cryosystem attached to a FEI Helios NanoLab 650 scanning electron microscope (Dept. of Chemistry, 4D Labs, Simon Fraser University). The frozen sample was sublimed for five minutes at −80°C, after which a thin layer of platinum (10-nm thickness) was sputter-coated onto the sample for 30 seconds at a current of 10 mA. The sample was moved into the SEM chamber, and the electron beam was set to a current of 50 pA at 3 kV. Images were captured at a working distance of 4 mm, at a scanning resolution of 3072 x 2207 collected over 128 low-dose scanning passes with drift correction.

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added. The original article lists references in alphabetical order; this version lists them by order of appearance, by design.