Canadian Journal of Plant Pathology

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tcjp20

First report of Fusarium proliferatum causing

crown and stem rot, and pith necrosis, in cannabis
(Cannabis sativa L., marijuana) plants

Zamir K. Punja

To cite this article: Zamir K. Punja (2020): First report of Fusarium�proliferatum causing crown and
stem rot, and pith necrosis, in cannabis (Cannabis�sativa L., marijuana) plants, Canadian Journal of
Plant Pathology, DOI: 10.1080/07060661.2020.1793222

To link to this article: https://doi.org/10.1080/07060661.2020.1793222

© 2020 The Author(s). Published by Informa

UK Limited, trading as Taylor & Francis
Group.

Published online: 06 Aug 2020.

Submit your article to this journal

Article views: 53

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tcjp20
Can. J. Plant Pathol., 2020
https://doi.org/10.1080/07060661.2020.1793222

Subject category: Epidemiology/Épidémiologie

First report of Fusarium proliferatum causing crown and stem rot,

and pith necrosis, in cannabis (Cannabis sativa L., marijuana) plants

ZAMIR K. PUNJA

Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada

(Accepted 2 July 2020)

Abstract: Cannabis (Cannabis sativa L., marijuana) plants grown under greenhouse or controlled environments with symptoms of leaf
yellowing, leaf necrosis and defoliation were observed during 2018–2019. Additional symptoms included crown rot and internal browning or
blackening of the pith tissues. Stock (mother) plants as well as plants in the vegetative and flowering stages of 15 cannabis strains
(genotypes) were affected. In addition, damping-off symptoms were observed on rooted cuttings in propagation rooms. Isolations from
diseased tissues yielded predominantly Fusarium proliferatum, with some F. oxysporum also recovered. Phylogenetic analysis of sequences
from the translation elongation factor 1α (TEF-1 α) region of 29 isolates of F. proliferatum from eight licenced production facilities in three
provinces in Canada (British Columbia, Ontario and New Brunswick), and one cannabis production site in northern California, grouped
isolates from cannabis with a large clade of isolates from a wide range of other hosts in different geographic regions. Pathogenicity studies
confirmed the ability of F. proliferatum to cause symptoms of wilting, leaf and pith necrosis, and plant death on cuttings, rooted plants and
stock plants. Inoculated tomato and cucumber plants developed similar symptoms. Stem colonization was more extensive by F. proliferatum
compared to F. oxysporum on cannabis cuttings. Both grew optimally at 25°C on agar media although F. oxysporum grew faster than
F. proliferatum at all temperatures tested. The occurrence of F. proliferatum on cannabis plants has not been previously reported, adding to
recent reports of F. oxysporum and F. solani that cause similar symptoms on cannabis plants.

Keywords: crown rot, damping-off, Gibberella fujikuroi, marijuana, pith necrosis

Résumé: En 2018-2019, on a observé des plants de cannabis (Cannabis sativa L., marijuana) cultivés en serre ou dans des environnements
contrôlés affichant des symptômes de jaunissement et de nécrose des feuilles ainsi que de défoliation. D’autres symptômes incluaient la pourriture
du collet et le brunissement interne ou le noircissement de la moelle. Des plants mères, de même que des plants au stade végétatif et de la floraison
de 15 souches (génotypes) de cannabis étaient touchés. De plus, on a observé, dans les salles de propagation, des symptômes de fonte des semis
chez les boutures racinées. Des isolements de tissus infectés ont révélé la présence de Fusarium proliferatum, principalement, ainsi que, dans une
moindre mesure, de F. oxysporum. L’analyse phylogénétique des séquences de la région du facteur d’élongation de la traduction 1α (TEF-1 α) de 29
isolats de F. proliferatum provenant de 8 installations de production autorisées de 3 provinces canadiennes (Colombie-Britannique, Ontario et
Nouveau-Brunswick) et d’une installation du nord de la Californie a regroupé les isolats de cannabis dans un grand clade d’isolats provenant d’une
vaste gamme d’autres hôtes de différentes régions géographiques. Des études de pathogénicité ont confirmé la capacité de F. proliferatum de
provoquer les symptômes du flétrissement, de la nécrose des feuilles et de la moelle ainsi que de causer la mort chez les boutures, les plants racinés
et les plants mères. Des plants de tomate et de concombre inoculés ont développé des symptômes similaires. Chez les boutures de cannabis, la
colonization des tiges par F. proliferatum était plus extensive que celle résultant de F. oxysporum. Les deux ont crû de façon optimale à 25°C sur de
la gélose dextrosée à la pomme de terre, bien que F. oxysporum se soit développé plus rapidement que F. proliferatum, et ce, à toutes les
températures testées. L’occurrence de F. proliferatum sur les plants de cannabis n’avait pas été rapportée jusqu’à maintenant, ce qui s’ajoute aux
récents rapports traitant de F. oxysporum et de F. solani qui causent des symptômes analogues sur les plants de cannabis.

Mots clés: fonte des semis, Gibberella fujikuroi, marijuana, nécrose de la moelle, pourriture du collet

Correspondence to: Zamir K. Punja E-mail: punja@sfu.ca

© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-
nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built
upon in any way.

Published online 06 Aug 2020

Z. K. Punja
Introduction
Cultivation of cannabis (Cannabis sativa L., marijuana)
occurs mainly in greenhouses and controlled environment
facilities throughout Canada, although an increasing num­
ber of field sites are also being approved for production, in
accordance with Health Canada requirements (Health
Canada 2019). With the increasing cultivation area of can­
nabis plants, the incidence of previously unreported patho­
gens is growing. Identification of the pathogens that affect
cannabis plants and recognition of the associated symp­
toms is necessary in order for cannabis producers to be able
to identify and manage the increased prevalence of these
diseases. Currently, a number of pathogens infecting can­
nabis plants have been described in Canada. These include
pathogens infecting the inflorescences, causing bud rots,
such as Botrytis cinerea, Penicillium species, and several
Fusarium species (F. oxysporum, F. solani,
F. sporotrichioides) (Punja et al. 2019; Punja 2020b,
2020d). Pathogens which infect the foliage include the
powdery mildew pathogens (Punja 2018, 2020c). Finally,
the previously described root and crown-infecting patho­
gens include Fusarium oxysporum, F. solani, several
Pythium species (Punja and Rodriguez 2018; Punja et al.
2020; Punja 2020a), and Cylindrocarpon (Punja 2020e).
In this study, the occurrence of F. proliferatum causing
root and crown rot, and pith necrosis, of cannabis plants
is described for the first time. The pathogen was found to
be present in seven licenced production facilities in three
provinces in Canada, and in one cannabis production site
in northern California. The pathogen was observed to be
more aggressive in pathogenicity tests compared to the
recently described report of F. oxysporum (Punja 2020a),
causing root and crown rot, and caused disease at all
stages of cannabis production, from propagation to vege­
tative growth to flowering, as well as infecting the
inflorescences. The progression of the pathogen into the
pith tissues following inoculation is demonstrated
through light and scanning electron microscopy.
Materials and methods
Sampling of plants
Cannabis plants showing symptoms of leaf yellowing,
stunted growth, and necrosis of leaves and browning of
roots, were included in the study. The plants were
sampled at various stages of development, ranging
from early propagation (1–2 weeks old) to vegetative
growth (3–4 weeks of age) to onset of full flowering
period (6–12 weeks of age) (Punja 2020a). They were
grown in greenhouses or in controlled environment
2
rooms in Health Canada approved licenced facilities.
Five were located in British Columbia, one in Ontario,
and one in New Brunswick. In addition, one production
site in northern California was included. The propagation
medium used was either coco fibre (coco coir) or rock­
wool blocks and plants were grown with the appropriate
nutrient regimes and lighting conditions as required for
commercial hydroponic production (Small 2017).
Microbial isolations were conducted during July 2018–
November 2019 and were made from over 200 sympto­
matic plants belonging to 15 cannabis strains (geno­
types). In addition, stock (mother) plants grown in
10 L containers (6–10 months of age) were included
for sampling (Table 1). The main stem of six diseased
stock plants (8–10 months of age) was cut down and
partitioned into 30-cm long increments, beginning at the
crown and proceeding alongside branches to the top of
the plant (>200 cm). From each increment, a 5-cm long
stem piece was dissected from the mid-point and sur­
face-sterilized as described below.
Isolation from tissues
Small pieces, ca. 0.5 cm in length of root segments or
0.2–0.4 cm2 for cuttings, stem pieces and bud tissues,
were surface-disinfested by dipping them in a 10% bleach
solution (0.625% NaOCl) for 1 min, followed by 30 s in
70% EtOH. The tissues were rinsed in sterile water and
blotted on paper towels. For larger diameter stem and pith
tissues, the sterilization protocol was increased to 2 min in
NaOCl and 30 s in EtOH. Tissue pieces were plated onto
potato dextrose agar (PDA, Sigma Chemicals, St. Louis,
MO) amended with 130 mg L−1 of streptomycin sulphate
(PDA+S). At least 20 pieces were plated from each sample,
four in each Petri dish. Dishes were incubated under ambi­
ent laboratory conditions (temperature range of 21–24°C
with 10–12 hr day−1 fluorescent lighting) for 5–10 days.
Emerging colonies were transferred to fresh PDA+S dishes
for subsequent identification to genus level using morpho­
logical criteria, including colony colour and size and micro­
scopic examination of spores. Up to 150 isolates putatively
identified as Fusarium spp. by morphological features as
described by Leslie and Summerell (2006) were obtained
from the different tissue types, among which both
F. oxysporum and F. proliferatum were then identified
using the PCR method described below. From these, 29
representative isolates of F. proliferatum were selected for
phylogenetic analysis to represent different tissue sources
and origins (Table 1). Colonies were transferred to water
agar and hyphal tip transfers were made onto PDA+S prior
to molecular analysis.
Crown rot on cannabis plants caused by Fusarium proliferatum 3

Table 1. Isolates of Fusarium proliferatum originating from cannabis (Cannabis sativa L., marijuana) plants included in this
study.
Isolate no. Source GenBank Submission no.

BC-1 Colony from air dispersal, around stock plants, on PDA MN784794
BC-2 Colony from air dispersal, around stock plants, on PDA -
BC-3 Colony from air dispersal, around vegetative plants, on PDA MN784793
BC-4 Colony from air dispersal, around vegetative plants, on PDA -
BC-5 Stock plant, internal pith blackening, strain ‘Hash Plant’ (Fig. 1e) MN784792
BC-6 Stock plant, internal pith browning, strain ‘Hash Plant’ (Fig. 1g) MN784791
BC-7 Stock plant, dead, crown isolation, strain ‘Pink Kush’ (Fig. 1c) MN784790
BC-8 Stock plant, dead, stem isolation, 10 cm height, strain ‘Hash Plant’ -
BC-9 Stock plant, dead, stem isolation, 20 cm height, strain ‘Hash Plant’ -
BC-10 Stock plant, dead stem, 100 cm height, strain ‘Hash Plant’ (Fig. 1g) -
BC-11 Stock plant, dead, stem isolation, 200 cm height, strain ‘Hash Plant’ MN784789
BC-12 Cuttings with damping-off symptoms, strain ‘Hash Plant’ MN784801
BC-13 Flowering plant, brown roots, strain ‘Purple God’ MN784787
BC-14 Flowering plant, brown roots, strain ‘Island Honey’ MN784799
BC-15 Flowering plant, brown pith, yellowing, strain ‘Hash Plant’ (Fig. 2c) MN784802
BC-16 Flowering plant, brown pith, yellowing, strain ‘Island Honey’ MN784803
BC-17 Flowering plant, brown pith, yellowing, strain ‘White Rhino’ (Fig. 2d) -
BC-18 Stock plant, crown isolation, strain ‘Blue Dream’ MN784788
BC-19 Stock plant, crown isolation, strain ‘Bubba’ MN784786
BC-20 Stock plant, crown isolation, strain ‘Lemon ZKittle’ MN784785
BC-21 Flower bud, harvested, strain ‘Purple God’ MN784804
BC-22 Flower bud, harvested, strain ‘Island Honey’ MN784805
BC-23 Flower bud stem base, strain ‘Purple God’ MN784806
BC-24 Stock plant, crown, strain ‘Emeral’ MN784807
BJ-1 Cuttings with damping-off symptoms, strain ‘Chem Dawg’ MT036760
NB-2 Cuttings with damping-off symptoms, strain ‘Sweet Durga’ MT036761
ON-1 Flowering plant, crown tissues, strain ‘White Shark’ MN784809
CA-1 Stock plant, brown roots, strain ‘Blueberry OG’ MN784814
CA-2 Stock plant, brown roots, strain ‘Sour Diesel’ MN784815
CA-3 Vegetative plant, roots, strain ‘Bubba Kush’ -

Isolates of Fusarium proliferatum were recovered during July 2018-November 2019 and originated from eight licenced production (LP) facilities.
Five were located in BC, one each in Ontario and New Brunswick, and one in northern California. Isolates BC-1 to BC-17, and BC-21 to BC-23, were
from one location (LP1), BC-18 to BC-19 were from a second location (LP2), BC-20 and BC-24 were from a third and fourth location (LP3 and LP4),
and BJ-1 was from a fifth LP (LP5). In addition, isolate ON-1 was from Ontario, isolate NB-2 was from New Brunswick, and isolates CA-1, CA-2,
CA-3 were from one site in northern California.
Sequences of isolates in bold have been submitted to GenBank. Corresponding numbers are provided.

Molecular identification uL 10 mM forward and reverse primers, as well as 20.25

Species-level identification of Fusarium isolates was
conducted using PCR with primers for the translation
elongation factor 1α (TEF-1 α) region: EF- 1 (5ʹATG
GGT AAG GAG GAC AAG AC 3ʹ) and EF-2 (5ʹ GGA
GGT ACC AGT GAT CAT GTT 3ʹ) (O’Donnell et al.
1998). Cultures of representative isolates from different
tissue sources (Table 1) were grown on potato dextrose
agar at room temperature for 7 days and DNA was
extracted from harvested mycelium scraped from the
colony surface using the QIAGEN DNeasy Plant Mini
Kit. Aliquots of 1 uL containing 5–20 ng DNA was used
for PCR in a 25 uL reaction volume consisting of 2.5 uL
10X buffer (containing 15 mM MgCl2), 0.5 uL 10 mM
dNTP, 0.25 uL Taq DNA Polymerase (QIAGEN), 0.25
uL DNAse- and RNAse-free water (Invitrogen). All PCR
amplifications were performed in a MyCycler thermo­
cycler (BIORAD) with the following program: 3 min at
94°C; 30 s at 94°C, 30 s at 60°C, 3 min at 72°C (35
cycles); and 7 min at 72°C. PCR products were separated
on 1% agarose gels and bands of the expected size (ca.
700 bp) were purified with QIAquick Gel Extraction Kit
and sent to Eurofins Genomics (Eurofins MWG Operon
LLC 2016, Louisville, KY) for sequencing. The resulting
sequences were compared to the corresponding EF-1 α
sequences from the National Centre for Biotechnology
Information (NCBI) GenBank database. Multiple
sequence alignment of the respective isolates was done
using the CLUSTAL W program (http://www.genome.jp/
tools/clustalw). The sequences of F. proliferatum were
Z. K. Punja
subsequently included in a phylogenetic analysis using
the neighbour-joining (NJ) method and a bootstrap con­
sensus tree was inferred from 1000 replicates as
described previously (Punja and Rodriguez 2018).
Effect of temperature on radial growth
To assess the effects of temperature on colony growth, 9-cm
diameter Petri dishes containing 25 mL of PDA were inocu­
lated with a 5 mm2 mycelial plug taken from one-week old
cultures of F. proliferatum originating from pith tissues
(isolate BC-5) and F. oxysporum (isolate BC-4). Replicates
of five Petri dishes were placed in temperature controlled
incubators maintained at 5, 10, 15, 20, 25, 30 and 35°C. The
actual observed temperatures were within ± 1°C. The dia­
meters of all colonies were measured after 6 days of incuba­
tion from two perpendicular measurements of each colony
and averaged. The experiment was conducted twice and the
data averaged.
Pathogenicity studies
Test-tube inoculations. A cotton plug was placed in the
bottom of 18 × 2.5-cm glass test-tubes and moistened
with sterile distilled water. A mycelial plug (8-mm dia­
meter) from 7-day old PDA cultures of either
F. proliferatum or F. oxysporum was placed on top of
the cotton plug, mycelial side facing up. The pathogeni­
city of an isolate of F. proliferatum obtained from dis­
eased pith tissues (isolate BC-5, Table 1) was compared
to isolate BC-4 of F. oxysporum from damped-off cut­
tings (Punja 2020a). A stem cutting (approx. 15 cm in
height) was then inserted into the test-tube to ensure
contact was made between the base of the cutting and
the mycelial plug. Control tubes received a PDA plug.
The test-tubes were sealed with caps and placed upright
in a test-tube rack for 7–10 days under ambient condi­
tions. After this time, the extent of mycelial development
on the stem cutting was measured with a ruler. Five
cannabis strains were assessed: ‘OG Kush’, ‘Hash
Plant’, ‘Copenhagen Kush’, ‘White Rhino’ and ‘Island
Honey’. There were four replicates of each strain and
isolate and the experiment was conducted twice. The
data from both experiments were averaged and variation
was expressed as standard errors.
Stem cutting inoculations. For inoculation of stem cut­
tings, the base of stem segments (15-cm height) from stock
plants of strains ‘Hash Plant’ and ‘White Rhino’ were
dipped into a spore suspension of F. proliferatum for
5 min and inserted into pre-moistened 4 cm2 rockwool
4
cubes (Grodan). The isolate was previously grown on
PDA plates for 7–10 days under ambient laboratory condi­
tions. The plates were flooded with 10 mL of sterile dis­
tilled water, the colony surface was rubbed with a glass rod,
and the resulting spore suspension was poured through two
layers of cheesecloth. Water was added as required to
achieve a concentration of 1 × 106 spores mL−1 as quanti­
fied in a haemocytometer. All rockwool cubes containing
inoculated cuttings were placed inside a plastic tray, a 1-cm
layer of water was added, and covered with a plastic dome
to maintain high humidity and placed in a Conviron incu­
bator set at 24°C with a 24-hr photoperiod for two weeks.
Controls consisted of noninoculated cuttings. For each
experimental trial, there were five replicate cuttings used.
Re-isolations were made from diseased tissues following
the surface-sterilization procedure described previously
and plating tissues onto PDA+S. The experiment was con­
ducted three times.
Rooted plant inoculation. Cuttings of strain ‘Hash Plant’
or ‘Critical Kali Mist’ were inserted into Jiffy-7® peat
pellets (http://www.jiffypot.com/) or rockwool cubes that
had been pre-soaked in Current Culture H2O® hydropo­
nic nutrient solution (https://www.cch2o.com/) to pro­
mote rooting (Punja and Rodriguez 2018). After two
weeks, the rooted cuttings were transferred to a coco
coir:perlite (3:1) potting medium in 10 cm2 pots. An
isolate of F. proliferatum originating from pith tissues
(BC-5) was grown in potato dextrose broth shake cul­
tures (100 mL) for 7–10 days at 150 rpm and the myce­
lial mat from two flasks was blended with 200 mL of
water for 20 s. To determine the inoculum level, dilu­
tions of the mycelial + spore suspension were made in
sterile distilled water (up to 10−3) and 50 uL was spread
onto the surface of PDA+S plates and incubated at
21–24°C for 5 days, at which time colony-forming
units (CFU mL−1) were quantified visually. Plants were
inoculated with 20 mL of the mycelial + spore suspen­
sion which was poured around the base of each plant.
A scalpel was then inserted into the soil at multiple
points around the roots to create wounds. The pots
were placed in a plastic tray containing a 1-cm layer of
water, and covered with a plastic dome to maintain high
humidity and placed in a Conviron incubator set at 24°C
with a 24-hr photoperiod. Symptoms of yellowing or
stunting of the plants were recorded after one and two
weeks. There were four replicate plants for each strain.
Re-isolations were made from diseased tissues following
the surface-sterilization procedure described previously
and plating tissues onto PDA+S. The experiment was
conducted twice.
Crown rot on cannabis plants caused by Fusarium proliferatum
Stock plant inoculations. Inoculations were made on
4 month old stock plants of strain ‘Space Queen’
grown in the coco-perlite mix by pouring 150 mL of
a mycelial + spore suspension prepared as described
above around the base of the plant. A scalpel was then
inserted into the soil at multiple points around the roots
to create wounds. The plants were placed in a Conviron
incubator set at 24°C with a 24-hr photoperiod.
Symptoms of yellowing, necrosis or stunting of the
plants were recorded after one and two weeks. Re-
isolations were made from diseased crown tissues fol­
lowing the surface-sterilization procedure described pre­
viously and plating tissues onto PDA+S. The experiment
was conducted twice.
Inoculation of tomato and cucumber plants. To deter­
mine whether F. proliferatum from cannabis plants could
infect tomato and cucumber plants, the following experi­
ments were conducted. Seeds of tomato ‘Moneymaker’
(West Coast Seeds) were planted in the coco:perlite mix
and plants grown for two weeks under 16-hr day−1
supplemental lighting at 23–26°C. The plants (replicated
six times) were then uprooted, roots washed under run­
ning tap water, and the bottom 4 cm was trimmed off
with a pair of scissors. The plants were immersed for
10 min in a mycelial + spore suspension prepared as
described above. Control plants (six replicates) had the
roots trimmed and were dipped in PDB broth. All plants
were re-potted and grown for an additional two weeks, at
which time symptoms of disease were recorded.
Cucumber seeds ‘Tasty Green’ (West Coast Seeds)
were grown for three weeks under the same conditions
as the tomato plants and were uprooted and had the roots
trimmed and dipped in inoculum for 10 min and re-
potted. Symptoms of disease were recorded after one
week. The experiments were conducted twice.
Light and scanning electron microscopy
Fungal samples. Colonies of F. proliferatum growing on
PDA+S for two weeks were used to examine spore
morphology. Small pieces of agar (3 mm2) with myce­
lium were adhered to an SEM 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 s to rapidly freeze it. After
freezing, the sample was placed in the preparation cham­
ber of a Quorum PP3010 T cryosystem attached to a FEI
Helios NanoLab 650 scanning electron microscope (4D
Labs., Simon Fraser University). The frozen sample was
5
sublimed for 5 min at −80°C, after which a thin layer of
platinum (10 nm thickness) was sputter-coated onto the
sample for 30 s 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 × 2207 collected over 128 low-dose scanning
passes with drift correction.
Pith tissue samples. Stem cuttings were taken from stock
plants of strains ‘Hash Plant’ and ‘Island Honey’. The
basal portion of the cutting was sliced into 5-mm long
sections which were each placed under a dissecting
microscope and photographed to show the internal struc­
ture and extent of development of the pith tissues. The
same stems were used to obtain similar slices which
were examined under the scanning electron microscope
as described above to show the cellular composition of
the pith cells and surrounding tissues. Stem sections
were also stained with a 1% solution of safranin red
for 30 min to show the lignin deposition within the
xylem tissues.
Inoculated cuttings. Stem cuttings were inoculated with
F. proliferatum by dipping them in a spore suspension as
described above and incubated at 24°C. Pieces of stem
segments (5 mm in length) that were showing early and
advanced stages of external colonization by mycelium
and with light brown external tissue discoloration (4–­
8 days after inoculation) were excised and examined
under the dissecting and scanning electron microscopes
as described above. Controls consisted of noninoculated
stem cuttings incubated under similar conditions.
Observations were made of the integrity of the pith
tissues and of parenchyma cells comprising the pith.
Results
Pathogen distribution in stock plants
Stock (mother) plants sampled in this study had reached
a height of 2–3 m and attained a basal stem diameter of
4–5 cm after 6–10 months of growth (Fig. 1a).
Symptoms of disease that affected some of the plants
(5–10% of the total) included necrosis of leaves and
defoliation (Fig. 1b). At advanced stages of disease
development, necrosis of leaves and stem death led to
plant mortality (Fig. 1c). At the crown region of many
diseased plants, the internal tissues were discoloured
(Fig. 1d). A black rot was visible internally around the
pith tissues when the plants were cut down (Fig. 1e). The
Z. K. Punja 6

Fig. 1 Symptoms of infection caused by Fusaium proliferatum on stock (mother plants). (a) Healthy plants approximately 6 months of age.
(b) Advanced infection causing yellowing and necrosis of leaves, and wilting symptoms. (c) Total collapse and defoliation of a severely
diseased plant. (d) Symptoms of crown decay in the plant shown in (b) with black streaks. (e) Cross-section through a stem of a stock plant
showing early internal black discoloration in the pith tissues. (f) More advanced internal stem discoloration and decay of the pith and xylem
tissues. (g) Pith decay resulting from infection by F. proliferatum at a distance of 100 cm from the crown. Stem on the far right is from
a healthy plant. (h) More advanced pith decay showing black streaks in the pith and shredding of the tissues. (i) Colonies of F. proliferatum
recovered from diseased pith tissues shown in (g).
Crown rot on cannabis plants caused by Fusarium proliferatum
internal discoloration was present in the stem at dis­
tances of 100–150 cm from the crown (Fig. 1f). At
advanced stages of disease development, the pith and
surrounding tissues were shredded (Fig. 1g, h). From
surface-sterilized crown and stem tissues of diseased
plants, colonies of Fusarium proliferatum (identified as
described below) were recovered at a frequency of
90–100% from the crown region (Fig. 1i). The colonies
produced fluffy aerial mycelium on PDA and developed
an intense purple pigment when viewed from the under­
side (Fig. 2a-d). A range of colony pigmentation and
growth rates were observed among isolates originating
from different sample sources (Table 1) which were all
identified as F. proliferatum. Under the scanning electron
microscope, microconidia were observed to be produced
in large numbers on PDA cultures, both in false heads at
the ends of phialides as well as in long chains (Fig. 2f-h).
Development of disease symptoms
Plants showing symptoms from which F. proliferatum
was isolated included rooted vegetative cuttings growing
in coco blocks displaying extensive necrosis of leaves
and plant collapse (Fig. 3a). Symptoms on flowering
plants included yellowing of leaves, browning of the
tips and margins of leaves, and wilting (Fig. 3b-d).
When the stems of these symptomatic plants were cut
open, extensive decay of the pith and surrounding tissues
was observed, which extended to distances of 30–50 cm
from the crown region (Fig. 3e, f). Asymptomatic cut­
tings taken from 100–150 cm distance away from the
crown of stock plants and inserted into PDA showed
presence of F. proliferatum at a frequency of around
1% after 7 days (Fig. 3g).
Molecular identification and phylogenetic analysis
Isolations made from around 200 symptomatic plants
representing 15 cannabis strains (genotypes) resulted in
over 300 isolates of Fusarium spp. (identified by mor­
phological features as described by Leslie and
Summerell 2006) being recovered, of which 150 were
subcultured and retained. These isolates were examined
for colony characteristics, pigmentation and spore pro­
duction using the morphological features shown in Fig. 2
to attempt to distinguish F. proliferatum from
F. oxysporum which was also recovered from these tis­
sues. Subsequently, PCR of the TEF-1 α region was
conducted to yield a band size of approximately 700 bp
(Fig. 4) in all isolates. Sequence analysis showed that
among the 150 isolates, 105 were F. proliferatum and 45
7
were F. oxysporum. A subset of 29 isolates of
F. proliferatum (Table 1) were included in the phyloge­
netic analysis. They included isolates recovered from 15
cannabis strains (genotypes) from eight licenced produc­
tion facilities in three provinces in Canada, and from one
cannabis production site in northern California. The tis­
sues from which the isolates were obtained included
diseased crowns and stem and pith tissues of stock plants
and flowering plants, damped-off cuttings, brown roots
on flowering plants, and infected flower buds (Table 1).
A few isolates were also recovered from air sampling
conducted by exposing Petri dishes in the growing envir­
onment for 60 min as described by Punja (2020a). The
phylogenetic analysis (Fig. 5) grouped isolates of
F. proliferatum from cannabis within a large clade of
isolates originating from a wide range of other hosts in
different geographical locations worldwide. There was
limited genetic variability among isolates representing
the species based on EF-1 sequences. Isolates of
F. oxysporum and F. solani previously isolated from
cannabis (Punja and Rodriguez 2018) were clearly sepa­
rated from F. proliferatum (Fig. 5).
Effect of temperature on radial growth
A comparison of the extent of colony growth of an isolate
each of F. proliferatum and F. oxysporum on PDA at seven
temperatures is shown in Fig. 6a. The optimal temperature
for radial growth of both species was 25°C, and growth was
reduced at 5–10°C but still occurred at 35°C. At all tem­
peratures tested, F. oxysporum grew faster than
F. proliferatum and colony diameters after 6 days at 25°C
were 72 mm and 50 mm, respectively (Fig. 6a). When
viewed from the underside, colonies grown at all tempera­
tures showed a pink-purple pigmentation, which was most
pronounced at 20, 25 and 30°C (Fig. 6b).
Pathogenicity studies
In the test-tube inoculation method using cuttings, myce­
lial growth of F. proliferatum was extensive after 7 days
on ‘Hash Plant’ (Fig. 7a), and infected cuttings removed
from the test-tube showed necrosis and wilting of the
leaves (Fig. 7b). Inoculation of cuttings of ‘White Rhino’
grown in rockwool cubes showed similar extensive
growth of mycelium on the stems, resulting in wilting
of the plant (Fig. 7c). In a coco:vermiculite growing
medium, inoculation of cuttings of ‘Hash Plant’ with
an inoculum level of 1 × 106 CFU mL−1 showed myce­
lial growth developing on the stem and initial wilting
was observed after 5 days (Fig. 7d), with more extensive
Z. K. Punja 8

Fig. 2 Recovery of Fusarium proliferatum from diseased stem tissues and growth characteristics on potato dextrose agar. (a) Surface view
(left) and bottom view (right) of isolation plates showing 100% recovery of F. proliferatum from diseased pith tissues after 7 days. (b) A one-
month-old isolation plate showing fluffy white aerial mycelium. (c, d) Six week-old colonies with white aerial mycelial strands on the colony
surface (c) and intense purple pigmentation produced on the underside of the colonies (d). (e) Comparison of the growth of six isolates of
F. proliferatum on PDA after 10 days. (f-h) Scanning electron micrographs of mycelium and spore production by F. proliferatum in culture.
(f) Microconidia produced in false heads. (g, h) Microconidia produced in long chains.
Crown rot on cannabis plants caused by Fusarium proliferatum 9

Fig. 3 Development of symptoms caused by Fusarium proliferatum on cannabis plants in the vegetative and flowering stages. (a) Foliar
necrosis and collapse of rooted cuttings. (b) Marginal necrosis of leaves. (c, d) Extensive yellowing and necrosis of foliage of flowering
plants of strains ‘Hash Plant’ and ‘White Rhino’. (e, f) Internal stem necrosis of the pith and xylem tissues of flowering plants shown in (c)
and (d). (g) Recovery of F. proliferatum from cuttings taken 100 cm from the crown of diseased plants and inserted into PDA slants.
Mycelial growth was visible after 5 days.

mycelial development and yellowing and wilting of the
plants occurring within 8 days (Fig. 7e). Noninoculated
control plants showed no symptoms (Fig. 7f). On rooted
inoculated cannabis plants of strain ‘Critical Kali Mist’,
symptoms of leaf necrosis and wilting of plants were
observed within 7–10 days (Fig. 7g). On inoculated
tomato and cucumber plants, disease symptoms were
severe and included total plant collapse and necrosis of
leaves within one week on cucumber plants and within
two weeks for tomato (Fig. 7h, i). On larger cannabis
Z. K. Punja 10

Fig. 4 PCR band of size ca. 700 bp obtained for 20 isolates of Fusarium proliferatum from cannabis plants (BC-5, BC-6, BC-8, BC-9, BC-
10, BC-11, BC-12, BC-13, BC-14, BC-15, BC-17, BC-18, BC-21, BC-22, BC-24, BJ-1, NB-2, ON-1, CA-1, CA-2) using the EF-1α primer
set. Lane C is the water control. Molecular weight standard is shown (L).

Fig. 5 Phylogenetic analysis of 23 isolates of Fusarium proliferatum originating from cannabis plants (see Table 1) using EF-1α sequences
compared to isolates from other hosts (GenBank numbers are shown). Isolates were obtained from a range of tissue sources and from
different licenced facilities in BC, ON, and NB, and one site in CA. A bootstrap consensus tree was inferred from 1000 replicates to
represent the distance using the neighbour-joining (NJ) method. Branches corresponding to partitions reproduced in less than 50% bootstrap
replicates were collapsed. The outgroup was Sclerotinia sclerotiorum.

stock plants of strain ‘Space Queen’, symptoms of foliar
necrosis developed on lower leaves within 5 days (Fig.
7j), and entire branches showed necrosis and wilting
after two weeks (Fig. 7k). The crown of affected plants
was discoloured and mycelial growth was evident in the
pith tissues further up the stem (Fig. 7l). Isolations made
from diseased plants yielded colonies of F. proliferatum.
Using the test-tube inoculation method to compare the
pathogenicity of F. proliferatum and F. oxysporum on
five cannabis strains, the results showed that stem
Crown rot on cannabis plants caused by Fusarium proliferatum 11

Fig. 6 (a) Comparison of radial growth of an isolate of Fusarium proliferatum (BC-5) with F. oxysporum (BC-4) at seven temperatures after
6 days. Means from five replicate dishes ± standard error are shown. The experiment was conducted twice. (b) Comparison of growth of
F. proliferatum after 6 days at 10–35°C, at 5°C increments. The underside of the colonies are shown to reveal the extent of pigmentation produced.

colonization was more extensive by F. proliferatum in
‘Hash Plant’, ‘Copenhagen Kush’ and ‘Island Honey’
and similar to F. oxysporum in ‘OG Kush’ and ‘White
Rhino’ (Fig. 8). All cannabis strains were considered to
be susceptible to both Fusarium species, with
F. proliferatum appearing to be more pathogenic on
some strains.
Light and scanning electron microscopy of pith tissues
Sections made through the stems of cuttings of cannabis
plants, when examined under both dissecting and scanning
electron microscopes, revealed the developmental progres­
sion of the pith tissues (Fig. 9). The cuttings showed
a progression from larger diameter pith areas with densely
Z. K. Punja 12

Fig. 7 Pathogenicity testing of Fusarium proliferatum using three different methods. (a) Stem cuttings of ‘Hash Plant’ were inoculated in a test-tube
with a mycelial plug. Mycelial growth on the stem is shown after 7 days of incubation. (b) Cuttings removed from the test-tubes to show mycelial
colonization of the stem and wilting of the cuttings. (c) Inoculation of cuttings of ‘Hash Plant’ by dipping in a spore suspension for 5 min and inserting
into a rockwool block. Stem colonization and wilting can be seen after 7 days. (d, e) Inoculation of rooted cuttings of ‘Hash Plant’ grown in a coco
coir: perlite potting mixture with a mycelial+spore suspension. (d) Wilting symptoms after 7 days. (e) Stem colonization and plant death after
two weeks. (f) Non-inoculated control plant shows no symptoms after two weeks. (g) Symptoms on ‘Critical Kali Mist’ plants two weeks after
inoculation with a mycelial + spore suspension. Extensive wilting and foliar necrosis can be seen on inoculated plant (right). (h) Symptoms on
a tomato plant inoculated with F. proliferatum (right) compared to the non-inoculated control plant (left) after two weeks. (i) Symptoms on a cucumber
plant inoculated with F. proliferatum (right) compared to the non-inoculated control plant (left) after two weeks. (j) Symptoms of foliar necrosis
developing on the lower part of a stock plant inoculated with a mycelial + spore suspension of F. proliferatum after one week. (k) The same plant after
two weeks showing stem die-back and wilting. (l) Decay of the crown tissues and mycelial growth inside the stem of the diseased plant shown in (k).
Crown rot on cannabis plants caused by Fusarium proliferatum 13

Fig. 8 Pathogenicity testing and response of five cannabis strains to inoculation with Fusarium oxysporum and F. proliferatum in a test-tube
assay. The extent of stem colonization was measured after 7 and 10 days. Data shown are for 10 days and are means of four replicates ±
standard errors. The experiment was conducted twice. Strains tested in each test-tube were: ‘OG Kush’, ‘Hash Plant’, ‘Copenhagen Kush’,
‘White Rhino’ and ‘Island Honey’. While there were no differences among strains in response to the two Fusarium species, colonization by
F. proliferatum was significantly greater on ‘Hash Plant’, ‘Copenhagen Kush’, and ‘Island Honey’ when compared to F. oxysporum.

packed pith cells (Fig. 9a, b) to smaller diameter pith areas
with loosely packed cells (Fig. 9c, d), to pith regions in
which large hollow centres developed (Fig. 9e-h). The
development of xylem vessel elements and tracheids can
be seen in Fig. 9d, f. Scanning electron microscopy of
a healthy stem cutting taken from a stock plant showed the
hollow pith surrounded by a ring of xylem tissues (Fig. 10a).
Healthy stem sections that were stained with 1% safranin red
showed the uptake of the red dye in the ring of xylem cells
(Fig. 10b). Scanning electron microscopy of a section made
through a young cannabis stem cutting showed the organiza­
tion of the outermost epidermal cell layer, with a dense
covering of hair-like trichomes, the inner ring of xylem
cells, and the innermost central parenchyma pith cells. The
cutting was inoculated with a spore suspension and the
mycelium progressed into the pith within 5 days after inocu­
lation. In contrast to non-inoculated stem cuttings, in which
the parenchyma cells surrounding the pith region were nor­
mal and the tissues were green (Fig. 11a, c), inoculated stem
tissues showed disruption of these tissues and intense
browning developed (Fig. 11a, c). When cells were exam­
ined by scanning electron microscopy, they had collapsed
and there was a build-up of a dense extracellular matrix that
could be a combination of cellular contents and polysacchar­
ides (Fig. 11d) compared to healthy cells (Fig. 11b).
Discussion
The isolation of F. proliferatum (Matsushima)
Nirenberg (Gibberella intermedia Kuhlman) from
a range of symptomatic tissues of cannabis plants, as
described in this study, adds to the previously
described reports of F. oxysporum and F. solani caus­
ing root and crown rot (Punja and Rodriguez 2018;
Punja 2020a), as well as F. brachygibbossum causing
crown infection on field-grown plants (Punja et al.
2018). The overlapping symptoms that develop fol­
lowing infection by these four pathogens requires
that isolation and pathogenicity studies be conducted
to confirm which species are present. In this study,
F. oxysporum was frequently recovered in conjunction
with F. proliferatum. The pathogenicity of isolates of
F. oxysporum has been confirmed in a previous study
(Punja 2020a). While F. proliferatum produces more
white aerial mycelium, forms spores in chains and in
false heads, develops a more intense purple pigment
on the underside of colonies on PDA, and grows more
slowly than F. oxysporum, PCR of the EF-1 region
was required to conclusively confirm the identity of
the isolates. Additional molecular approaches used to
distinguish between these two Fusarium species have
included the use of calmodulin gene sequences (Mule
et al. 2004; Chang et al. 2015), the internal transcribed
spacer region of rDNA (Quazi 2013), and the inter­
generic spacer region of rDNA (Palmero et al. 2012).
The optimal temperature for growth of F. proliferatum
was 25°C, confirming results from an earlier study
(Marin et al. 1995). The growth of F. proliferatum
was slower than that of F. oxysporum but it was
more pathogenic on cannabis stem cuttings.
The epidemiology of F. proliferatum is quite similar to that
of F. oxysporum (Punja 2020a), and the pathogen was recov­
ered from cuttings, vegetative and flowering plants, and
stock plants, and was aerially dispersed and also recovered
Z. K. Punja 14

Fig. 9 The extent of development of the central pith tissues in cannabis stem cuttings. The cuttings were obtained from stock plants at the
same stage of growth and sequential stem pieces were taken (from top to bottom) and sectioned by hand and 3 mm thick slices were placed
on the observation stage of a dissecting microscope and photographed at the same magnification. (a, b) Early stage of pith development with
densely packed central parenchyma cells. (c, d) Loosely packed parenchyma cells in the pith region. (e, f) Development of hollow central
pith where parenchyma cells are absent. (g, h) Complete development of central pith in stem cutting of cannabis strain ‘Hash Plant’. Scale
bars shown in a and b apply to all figures.
Crown rot on cannabis plants caused by Fusarium proliferatum 15

Fig. 10 (a) Scanning electron micrograph of a healthy stem cutting taken from a stock plant showing the hollow pith surrounded by a ring of
xylem tissues. A portion of the remaining pith cells (arrow) that has not disintegrated can be seen. (b) Healthy stem sections that were stained
with 1% safranin red to show the uptake of the red dye in the ring of xylem cells (arrow). (c) Scanning electron micrograph of a section made
through a young cannabis stem cutting to show the organization of the outermost epidermal cell layer with a dense covering of hair-like
trichomes, the inner ring of xylem cells, and the innermost central parenchyma pith cells. The cutting was inoculated with a spore suspension
of Fusarium proliferatum and the mycelium has progressed into the pith (white arrow). The advancement of the mycelial front is shown by
the blue arrows. Photo was taken 5 days after inoculation.
Z. K. Punja 16

Fig. 11 Comparison of hand sections made through a non-inoculated stem cutting with those infected by F. proliferatum. (a) Healthy cutting
(top left) compared with three different stages of necrosis and tissue disruption caused by pathogen invasion of inoculated stem cuttings. (b)
Healthy parenchyma cells viewed under the scanning electron microscope. (c) Close-up of a healthy cutting (left) with a diseased cutting
(right). (d) Parenchyma cells in a diseased cutting that are disrupted and show accumulation of a dense matrix that could be a combination of
cellular contents and polysaccharides.

from infected inflorescences. The primary differences in
symptomology between the two pathogens were that
F. proliferatum caused a distinct browning/blackening within
the pith tissues of affected plants and foliar necrosis was
more noticeable compared to yellowing of leaves caused
by F. oxysporum (Punja 2020a). The pith tissues of plants
are prone to infection by a range of pathogens affecting
a number of different hosts. Visible browning of the pith
tissues similar to that observed on cannabis plants in this
study has been reported in several other diseases e.g. black
shank of tobacco (Phytophthora nicotianae) (Gallup et al.
2006), brown stem rot of soybeans (Phialophora gregata)
(Hadi 2016), fusarium stalk rot of corn (Fusarium monili­
forme) (Faske and Kirkpatrick 2015), and pith necrosis of
tomato (Pseudomonas corrugata) (Scarlett et al. 2015) and
Xanthomonas perforans (Aiello et al. 2013). The pith tissues
consist of loosely organized parenchyma cells that store and
transport nutrients (Fujimoto et al. 2018). The morphological
features of parenchyma cells and pith development in canna­
bis stem tissues, as shown by light and scanning electron
microscopy in this study, have not been previously described.
During the development of a central pith in plants such as
sorghum, corn and rice, death of the cells in the stem pith
parenchyma occurs in a manner similar to that of pro­
grammed cell death (PCD) (Beers 1997; Fujimoto et al.
2018). Activation of autolytic cell wall degrading enzymes
resulted in the development of a central cavity (Beers 1997;
Fujimoto et al. 2018). In sorghum, this cell death resulted in
increased susceptibility to the anthracnose pathogen,
Colletotrichum graminicola (Katsanos and Pappelis 1966).
It is not clear whether pathogen spores and/or pathogen
metabolites can spread within the pith tissues of cannabis
plants but recovery of the pathogen at distances of
100–150 cm from the crown suggest that long-range spread
internally of F. proliferatum is occurring in diseased plants,
similar to that reported for F. oxysporum (Punja 2020a). The
rapidity of symptom development on inoculated cannabis
plants and the development of necrosis and wilting symp­
toms also suggest that pathogen metabolites are a part of the
disease syndrome. SEM images of the pith tissues from
inoculated cuttings showed collapse of parenchyma cells
and cell death within 5 days, and was accompanied by an
accumulation of a dense matrix in diseased pith tissues, likely
due to an accumulation of polysaccharides and the result of
Crown rot on cannabis plants caused by Fusarium proliferatum
host cell wall destruction. Mycelial growth was observed
ramifying from the epidermal cells into the pith tissues.
Fusarium proliferatum is a member of the Gibberella
fujikuroi species complex, which is comprised at least of 15
reproductively isolated biological species (mating popula­
tions), and infects a broad range of plant species and
produces a range of mycotoxins (Leslie et al. 2004;
Niehaus et al. 2016). Some of the more economically
important host species affected by F. proliferatum are
asparagus (Elmer 1990, 1995; Borrego-Benjumea et al.
2014), alfalfa (Cong et al. 2016), banana (Jimenez et al.
1993), citrus fruits (Hyun et al. 2000), corn (Logrieco et al.
1995; Munkvold 2003), garlic (Dugan et al. 2003; Palmero
et al. 2012; Galvez et al. 2017), mango (Zhan et al. 2010),
onion (Stankovic et al. 2007; Carrieri et al. 2013), orchids
(Benyon et al. 1996), peach palm (Jarek et al. 2018),
pistachio (Crespo et al. 2019), rice (Desjardins et al.
2000; Kim et al. 2012; Quazi et al. 2013; Choi et al.
2017, 2018), safflower (Kim et al. 2016), sesame (Torabi
et al. 2014), soybean (Chang et al. 2015), sorghum (Leslie
2003), strawberry (Borrero et al. 2019), and wheat (Jurado
et al. 2006; Conner et al. 2009). Symptoms on these hosts
included damping-off, wilting, yellowing of leaves, soft
rot, root and crown rot, vascular discoloration, as well as
blackening of the crown and roots of diseased plants as
seen on palm and strawberry plants (Jarek et al. 2018;
Borrero et al. 2019). All of these symptoms were observed
on diseased cannabis plants affected by F. proliferatum in
this study.
The mycotoxins reported to be produced by
F. proliferatum include the polyketide-derived fumoni­
sins, with fumonisin B1 (FB1) being the most prevalent
(Rheeder et al. 2002; Jurado et al. 2010; Stepien et al.
2011; Choi et al. 2017; Galvez et al. 2017). In addition, the
following mycotoxins – beauvericin, enniatin, fusaric
acid, fusarin, fusaproliferin, and moniliformin – are also
produced by F. proliferatum (Marasas et al. 1986; Ritieni
et al. 1995; Moretti et al. 1996; Bacon et al. 1996;
Desjardins et al. 2000; Logrieco et al. 2002; Leslie et al.
2004; Desjardins 2006; Proctor et al. 2010; Niehaus et al.
2016; Galvez et al. 2017). It is not known if any of these
metabolites are produced in diseased cannabis tissues but
the infection of inflorescences by this pathogen, as
reported in this study and in a recent study (Punja
2020b), may be of concern. In addition, the occurrence
of F. sporotrichioides on diseased inflorescences (Punja
2020b) could also raise concern over the potential for
mycotoxin production. This species produces a range of
trichothecene mycotoxins, including T-2 toxin, neosola­
niol, nivalenol, and HT-2 toxin (Kokkonen et al. 2012). In
wheat plants, Guo et al. (2016) demonstrated that seed-
17
borne infection by F. proliferatum resulted in internal
systemic spread of the pathogen within the stem tissues,
which led to seed kernel infection and mycotoxin accu­
mulation (fuminosin and beauvericin). The potential for
systemic movement of Fusarium species through the pith
and xylem tissues of cannabis plants to result in infection
of the inflorescence needs to be investigated. This could
potentially occur given that pathogen recovery was con­
firmed in this study at distances as far as 100–150 cm from
the crown region. Previous studies also showed that
F. oxysporum demonstrated endophytic colonization of
stems (Punja et al. 2019) and was transmitted through
cuttings taken from symptomless cannabis plants (Punja
2020a). In corn, inoculation studies conducted with
a GFP-marked strain of Fusarium verticilliodes showed
that the pathogen, when applied at the seedling stage,
resulted in infection through the radicle which progressed
upward through the stem tissues to the ear to infect kernels
(Gai et al. 2018). The extent to which Fusarium spp. are
transmitted as seed-borne pathogens in cannabis plants is
not known but a similar infection cycle that involves
spread though the pith could potentially give rise to patho­
gen-contaminated seed.
In British Columbia, F. proliferatum has been previously
isolated from garlic bulbs and pine seedlings (Joshi and
Jeffries 2018). In other regions of Canada, it also causes
crown and root rot of asparagus (Vujanovic et al. 2006;
Borrego-Benjumea et al. 2014) and soybeans (Chang et al.
2015) and stalk rot of corn (Reid and Zhu 2004) and has
been detected in wheat seed (Clear and Patrick 1990).
Fusarium proliferatum has been reported to be more patho­
genic that F. oxysporum on soybeans (Chang et al. 2015),
an observation confirmed in this study on inoculated can­
nabis cuttings. The pathogen is also capable of extensive
colonization of organic matter in soil (Gaige et al. 2019).
Fusarium oxysporum and F. proliferatum are frequently
isolated from the same diseased plants, e.g. in asparagus
(Borrego-Benjumea et al. 2014), soybeans (Chang et al.
2015), and peach palm (Jarek et al. 2018), similar to what
was observed in this study. Dual inoculations with both
species were not conducted given the severe symptoms
produced by each individual species. The ability to produce
an array of metabolically active compounds could be
important in the infection potential of F. proliferatum on
other hosts (Niehaus et al. 2016). Both cucumber and
tomato plants inoculated in this study developed rapid
necrosis of the foliage, followed by plant death, which
may suggest a role for extracellular compounds in symp­
tom development. Fusaric acid, for example, has been
shown to be associated with symptom development in
plants affected by F. proliferatum (Palmero et al. 2012).
Z. K. Punja
The approaches to management of F. proliferatum
should be similar to those suggested for F. oxysporum
(Punja 2020a), given that both pathogens can occur
concurrently in diseased cannabis tissues despite the
different symptomologies. However, specific studies to
address the management of F. proliferatum have not yet
been conducted given the pathogen has not been pre­
viously described from cannabis. This represents the first
report of F. proliferatum causing root and crown rot, and
pith necrosis, on cannabis plants. The detection of this
pathogen in eight licenced production facilities in three
provinces in Canada (British Columbia, Ontario and
New Brunswick), and one cannabis production site in
northern California, suggests the pathogen may have
been present for some time and been spread through
diseased planting stock or through other means and
remained undetected. The recovery of the pathogen
from 15 cannabis strains suggests that identification of
genetic resistance may prove to be challenging unless
a broader spectrum of genotypes, which include land
races, are evaluated (Punja et al. 2017).
Acknowledgements
Technical assistance provided by Samantha Lung, Emily
Betz, Cameron Scott, Darren Sutton, Hayley Reekie, and
Sarah Chen during various aspects of this project, is
gratefully acknowledged. This work made use of the
4D LABS scanning electron microscopy facility sup­
ported by the Canada Foundation for Innovation (CFI),
British Columbia Knowledge Development Fund
(BCKDF), Western Economic Diversification Canada
(WD), and Simon Fraser University (SFU)
Funding
This research was funded through a financial contribu­
tion from Agrima Botanicals and from matching funds in
a Collaborative Research and Development (CRD) Grant
from the Natural Sciences and Engineering Research
Council of Canada (NSERC).
References
Aiello D, Scuderi G, Vitale A, Firrao G, Polizzi G, Cirvilleri G. 2013.
A pith necrosis caused by Xanthomonas perforans on tomato plants. Eur
J Plant Pathol. 137(1):29–41. doi:10.1007/s10658-013-0214-7.
Bacon CW, Porter JK, Norred WP, Leslie JF. 1996. Production of
fusaric acid by Fusarium species. Appl Environ Microbiol.
62:4039–4043. doi:10.1128/AEM.62.11.4039-4043.1996.
Beers EP. 1997. Programmed cell death during plant growth and
development. Cell Death Differ. 4:649–661. doi:10.1038/sj.cdd.4400297.
18
Benyon F, Summerell BA, Burgess LW. 1996. Association of Fusarium
species with root rot of Cymbidium orchids. Australas Plant Path.
25:226–228. doi:10.1071/AP96041.
Borrego-Benjumea A, Basallote-Ureba MJ, Melero-Vara JM,
Abbasi PA. 2014. Characterization of Fusarium isolates from aspara­
gus fields in southwestern Ontario and influence of soil organic amend­
ments on Fusarium crown and root rot. Phytopathology. 104
(4):403–415. doi:10.1094/PHYTO-08-13-0231-R.
Borrero C, Capote N, Gallardo MA, Aviles M. 2019. First report of
vascular wilt caused by Fusarium proliferatum on strawberry in Spain.
Plant Dis. 103(3):581. doi:10.1094/PDIS-09-18-1544-PDN.
Carrieri R, Raimo F, Pentangelo A, Lahoz E. 2013. Fusarium prolif­
eratum and Fusarium tricinctum as causal agents of pink rot of onion
bulbs and the effect of soil solarization combined with compost amend­
ment in controlling their infections in field. Crop Prot. 43:31–37.
doi:10.1016/j.cropro.2012.09.013.
Chang KF, Hwang SF, Conner RL, Ahmed HU, Zhou Q,
Turnbull GD, Strelkov SE, McLaren DL, Gossen BD. 2015. First
report of Fusarium proliferatum causing root rot in soybean (Glycine max
L.) in Canada. Crop Prot. 67:52–58. doi:10.1016/j.cropro.2014.09.020.
Choi H-W, Hong SK, Lee YK, Kim WG, Chun S. 2018. Taxonomy of
Fusarium fujikuroi species complex associated with bakanae on rice in
Korea. Australas Plant Pathol. 47(1):23–34. doi:10.1007/s13313-017-
0536-6.
Choi JH, Lee S, Nah JY, Kim HK, Paek JS, Lee S, Ham H,
Hong SK, Yun SH, Lee T. 2017. Species composition of and fumo­
nisin production by the Fusarium fujikuroi species complex isolated
from Korean cereals. Intern. J Food Microbiol. 267:62–69.
doi:10.1016/j.ijfoodmicro.2017.12.006.
Clear RM, Patrick SK. 1990. Fusarium species isolated from wheat sam­
ples containing tomb-stone (scab) kernels from Ontario, Manitoba, and
Saskatchewan. Can J Plant Sci. 70:1057–1069. doi:10.4141/cjps90-128.
Cong LL, Sun Y, Kang JM, Li MN, Long RC, Zhang TJ, Yang QC.
2016. First report of root rot disease caused by Fusarium proliferatum
on alfalfa in China. Plant Dis. 100(12):25–26. doi:10.1094/PDIS-08-14-
0790-SR.
Conner RL, Hwang SF, Stevens RR. 2009. Fusarium proliferatum:
a new causal agent of black point in wheat. Can J Plant Pathol.
18:419–423. doi:10.1080/07060669609500598.
Crespo M, Lawrence DP, Nouri MT, Doll DA, Trouillas FP. 2019.
Characterization of Fusarium and Neocosmospora species associated
with crown rot and stem canker of pistachio rootstocks in California.
Plant Dis. 103(8):1931–1939. doi:10.1094/PDIS-11-18-2012-RE.
Desjardins AE. 2006. Fusarium mycotoxins: chemistry, genetics, and
biology. St. Paul (MN): APS Press.
Desjardins AE, Manandhar HK, Plattner RD, Manandhar GG,
Poling SM, Maragos CM. 2000. Fusarium species from Nepalese
rice and production of mycotoxins and gibberellic acid by selected
species. Appl Environ Microbiol. 66:1020–1025. doi:10.1128/
AEM.66.3.1020-1025.2000.
Dugan FM, Hellier BC, Lupien SL. 2003. First report of Fusarium
proliferatum causing rot of garlic bulbs in North America. Plant Pathol.
52:426. doi:10.1046/j.1365-3059.2003.00852.x.
Elmer WH. 1990. Fusarium proliferatum as a causal agent in Fusarium
crown and root rot of asparagus. Plant Dis. 74:938. doi:10.1094/PD-74-
0938E.
Elmer WH. 1995. A single mating population of Gibberella fujikuroi
(Fusarium proliferatum) predominates in asparagus fields in
Connecticut, Massachusetts, and Michigan. Mycologia. 87:68–71.
doi:10.1080/00275514.1995.12026504.
Faske T, Kirkpatrick T 2015. Corn diseases and nematodes. Chapter 7.
In Arkansas corn production handbook. University of Arkansas
Research and Extension. MP437. Accessed 2020 Feb 24. https://www.
uaex.edu/publications/pdf/mp437/chapter7corn.pdf.
Crown rot on cannabis plants caused by Fusarium proliferatum
Fujimoto M, Sazuka T, Oda Y, Kawahigashi H, Wu J, Takanashi H,
Ohnishi T, Yoneda J-I, Ishimori M, Kajiya-Kanegae H, et al.
2018. Transcriptional switch for programmed cell death in pith parench­
yma of sorghum cells. Proc Natl Acad Sci USA. 115(37):E8783–E8792.
doi:10.1073/pnas.1807501115
Gai X, Dong H, Wang S, Liu B, Zhang Z, Li X, Gao Z. 2018.
Infection cycle of maize stalk rot and ear rot caused by Fusarium
verticillioides. PLoS ONE. 13(7):e0201588. doi:10.1371/journal.
pone.0201588.
Gaige AR, Giraldo M, Todd T, Stack JP. 2019. Growth and coloniza­
tion of organic matter in soil by Fusarium proliferatum. Can J Plant
Pathol. 41:242–250. doi:10.1080/07060661.2018.1522374.
Gallup CA, Sullivan MJ, Shew HD. 2006. Black shank of tobacco.
American Phytopathological Society. The Plant Health Instructor.
doi:10.1094/PHI-I-2006-0717-01
Galvez L, Urbaniak M, Waskiewicz A, Srepien L, Palmero D. 2017.
Fusarium proliferatum – causal agent of garlic bulb rot in Spain: genetic
variability and mycotoxin production. Food Microbiol. 67:41–48.
doi:10.1016/j.fm.2017.05.006.
Guo Z, Pfohl K, Karlovsky P, Dehne H-W, Altincicek B. 2016.
Fuminosin B1 and beauvericin accumulation in wheat kernels after
seed-borne infection with Fusarium proliferatum. Agricul Food Sci.
25:138–145. doi:10.23986/afsci.55539.
Hadi B 2016. Brown stem rot of soybeans. Northern Plains Integrated Pest
Management Guide. Accessed 2020 Feb 14. https://wiki.bugwood.org/
NPIPM:Brown_stem_rot_of_soybean.
Health Canada. 2019. Accessed 2019 Sept 16. https://www.canada.ca/en/
health-canada/services/drugs-medication/cannabis/industry-licensees-
applicants/licensed-cultivators-processors-sellers.html.
Hyun JW, Lee SC, Kim DH, Ko SW, Kim KS. 2000. Fusarium fruit rot
of citrus in Jeju Island. Mycobiology. 28:158–162. doi:10.1080/
12298093.2000.12015743.
Jarek TM, Dos Santos AF, Tessmann DJ, Vieira ESN. 2018.
Inoculation methods and aggressiveness of five Fusarium species
against peach palm. Ciencia Rural. 48:04 e20170462. doi:10.1590/
0103-8478cr20170462.
Jimenez M, Logrieco A, Bottalico A. 1993. Occurrence and pathogeni­
city of Fusarium species in banana fruits. J. Phytopathol. 137
(3):214–220. doi:10.1111/j.1439-0434.1993.tb01341.x.
Joshi V, Jeffries M. 2018. Diseases/symptoms diagnosed on commercial
crop samples submitted to the British Columbia Ministry of Agriculture
(BCAGRI) Plant Health Laboratory in 2017. Can Plant Dis Survey.
98:6–16.
Jurado M, Marín P, Callejas C, Moretti A, Vázquez C, González-
Jaén MT. 2010. Genetic variability and fumonisin production by
Fusarium proliferatum. Food Microbiol. 27:50–57. doi:10.1016/j.
fm.2009.08.001.
Jurado M, Vázquez C, Callejas C, González Jaén MT. 2006.
Occurrence and variability of mycotoxigenic Fusarium species asso­
ciated to wheat and maize in the southwest of Spain. Mycotoxin Res.
22:87–91. doi:10.1007/BF02956769.
Katsanos RA, Pappelis AJ. 1966. Relationship of cell death patterns and
spread of Colletotrichum graminicola in sorghum stalk tissue.
Phytopathology. 56:468–469.
Kim J-H, Kang M-R, Kim H-K, Lee S-H, Lee T, Yun S-H. 2012.
Population structure of the Gibberella fujikuroi species complex asso­
ciated with rice and corn in Korea. Plant Pathol. J. 28(4):357–363.
doi:10.5423/PPJ.OA.09.2012.0134.
Kim SG, Ko H-C, Hur O-S, Luitel BP, Rhee J-H, Yoon M-S,
Baek H-J, Ryu K-Y, Sung JS. 2016. First report of Fusarium wilt
caused by Fusarium proliferatum on safflower. Res Plant Dis. 22
(2):111–115. doi:10.5423/RPD.2016.22.2.111.
19
Kokkonen M, Jestoi M, Laitila A. 2012. Mycotoxin production of
Fusarium langsethiae and Fusarium sporotrichioides on cereal-based
substrates. Mycotoxin Res. 28:25–35. doi:10.1007/s12550-011-0113-8.
Leslie JF. 2003. Sorghum and millet diseases. Ames (IA): Iowa State
Press.
Leslie JF, Summerell BA. 2006. The Fusarium laboratory manual.
Ames (IA): Blackwell.
Leslie JF, Zeller KA, Logrieco A, Mulè G, Moretti A, Ritieni A.
2004. Species diversity of and toxin production by Gibberella fujikuroi
species complex strains isolated from native prairie grasses in Kansas.
Appl Environ Microbiol. 70:2254–2262. doi:10.1128/AEM.70.4.2254-
2262.2004.
Logrieco A, Moretti A, Ritieni A, Bottalico A, Corda P. 1995.
Occurrence and toxigenicity of Fusarium proliferatum from preharvest
maize ear rot, and associated mycotoxins, in Italy. Plant Dis.
79:727–731. doi:10.1094/PD-79-0727.
Logrieco A, Mule G, Moretti A, Bottalico A. 2002. Toxigenic Fusarium
species and mycotoxins associated with maize ear rot in Europe. Eur J Plant
Pathol. 108:597–609. doi:10.1023/A:1020679029993.
Marasas WFO, Thiel PG, Rabie CJ, Nelson PE, Toussoun TA. 1986.
Moniliformin production in Fusarium section Liseola. Mycologia.
78:242–247. doi:10.1080/00275514.1986.12025235.
Marin S, Sanchis V, Magan N. 1995. Water activity, temperature, and
pH effects on growth of Fusarium moniliforme and Fusarium prolifer­
atum isolates from maize. Can J Microbiol. 41:1063–1070. doi:10.1139/
m95-149.
Moretti A, Logrieco A, Bottalico A, Ritieni A, Fogliano V,
Randazzo G. 1996. Diversity in beauvericin and fusaproliferin produc­
tion by different populations of Gibberella fujikuroi (Fusarium section
Liseola). Sydowia. 48:44–56.
Mule G, Susca A, Stea G, Moretti A. 2004. Specific detection of the
toxigenic species Fusarium proliferatum and F. oxysporum from aspar­
agus plants using primers based on calmodulin gene sequences. FEWS
Microbiol Lett. 230:235–240. doi:10.1016/S0378-1097(03)00926-1.
Munkvold GP. 2003. Epidemiology of mycotoxin producing fungi.
Netherlands: Springer.
Niehaus E-M, Munsterkotter M, Proctor RH, Brown DW, Sharon A,
Idan Y, Oren-Young L, Sieber CM, Novák O, Pěnčík A, et al.
2016. Comparative “Omics” of the Fusarium fujikuroi species complex
highlights differences in genetic potential and metabolite synthesis.
Genome Bio Evol. 8(11):3574–3599. doi:10.1093/gbe/evw259
O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC. 1998. Multiple
evolutionary origins of the fungus causing Panama disease of banana:
concordant evidence from nuclear and mitochondrial gene genealogies.
Proc Natl Acad Sci USA. 95:2044–2049. doi:10.1073/pnas.95.5.2044.
Palmero D, De Cara M, Nosir W, Galvez L, Cruz A, Woodward S,
Gonzalez-Jaen MT, Tello JC. 2012. Fusarium proliferatum isolated
from garlic in Spain: identification, toxigenic potential and pathogeni­
city on related Allium species. Phytopathol Mediter. 51:207–218.
Proctor RH, Desjardins AE, Moretti A. 2010. Biological and chemical
complexity of Fusarium proliferatum. In: Strange RN, Gullino ML,
editors. The role of plant pathology in food safety and food security. Plant
pathology in the 21st century. Netherlands: Springer Science; p. 97–111.
Punja ZK. 2018. Flower and foliage-infecting pathogens of marijuana
(Cannabis sativa L.) plants. Can J Plant Pathol. 40:514–527.
doi:10.1080/07060661.2018.1535467.
Punja ZK.2020a. Epidemiology of Fusarium oxysporum causing root and
crown rot of cannabis (Cannabis sativa L., marijuana) plants in com­
mercial greenhouse production. Can J. Plant Pathol. 42(3).(in press)
Punja ZK.2020b. The diverse mycoflora present on dried cannabis
(Cannabis sativa L.) inflorescences in commercial production. Can
J Plant Pathol. 42(3).(in press)
Z. K. Punja
Punja ZK. 2020c. First report of the hops powdery mildew pathogen,
Podosphaeria macularis, on naturally infected marijuana (Cannabis
sativa L.) plants in the field. Phytopathology (Abstr). (in press).
Punja ZK. 2020d. Cannabis and hemp biology and pathology: an overview
of the crop and emerging pathogens. Phytopathology (Abstr.). (in press).
Punja ZK. 2020e. Brown root rot and crown rot of cannabis (Cannabis
sativa L., marijuana) plants caused by Fusarium (Cylindrocarpon)
lichenicola. Phytopathology (Abstr.). (in press).
Punja ZK, Collyer D, Scott C, Lung S, Holmes J, Sutton D. 2019.
Pathogens and molds affecting production and quality of Cannabis
sativa L. Frontiers Plant Sci. (on-line). doi:10.3389/fpls.2019.01120.
Punja ZK, Rodriguez G. 2018. Fusarium and Pythium species infecting
roots of hydroponically grown marijuana (Cannabis sativa L.) plants. Can
J Plant Pathol. 40(4):498–513. doi:10.1080/07060661.2018.1535466.
Punja ZK, Rodriguez G, Chen S. 2017. Assessing genetic diversity in
Cannabis sativa using molecular approaches. In: Chandra S, Lata L,
ElSohly MA, editors. Cannabis sativa L. Botany and Biotechnology.
Berlin: Springer-Verlag; p. 395–418.
Punja ZK, Scott C, Chen S. 2018. Root and crown rot pathogens causing
wilt symptoms on field-grown marijuana (Cannabis sativa L.) plants. Can
J Plant Pathol. 40(4):528–541. doi:10.1080/07060661.2018.1535470.
Punja ZK, Scott C, Lung S, Roberts A. 2020. The Pythium species
complex associated with crown and root rot on cannabis (Cannabis
sativa L., marijuana) plants grown under commercial greenhouse
conditions. Phytopathology. (Abstr.). (in press).
Quazi SAJ, Meon S, Jaafar H, Ahmad ZABM. 2013. Characterization
of Fusarium proliferatum through species specific primers and its viru­
lence on rice seeds. Intern J Agric Biol. 15(4):649–656.
Reid LM, Zhu X 2004. Common diseases of silage corn in Canada. In
Chapt. 6: Corn Pests. Advanced Silage Corn Management 2004. Accessed
2020 Feb 17. Farwmwest.com. https://farmwest.com/node/966
20
Rheeder JP, Marasas WF, Vismer HF. 2002. Production of fumonisin
analogs by Fusarium species. Appl Environ Microb. 68:2101–2105.
doi:10.1128/AEM.68.5.2101-2105.2002.
Ritieni A, Fogliano V, Randazzo G, Scarallo A, Logrieco A,
Moretti A, Bottalico A, Mannina L. 1995. Isolation and character­
ization of fusaproliferin, a new toxic metabolite from Fusarium
proliferatum. Nat Toxins. 3:17–20. doi:10.1002/nt.2620030105.
Scarlett K, Tesoriero L, Daniel R, Maffi D, Faoro F, Guest DI. 2015.
Airborne inoculum of Fusarium oxysporum f. sp. cucumerinum. Eur J
Plant Pathol. 141:779–787. doi:10.1007/s10658-014-0578-3.
Small E. 2017. Cannabis. In: A Complete Guide. Boca Raton FL: CRC Press;
567 p.
Stankovic S, Levic J, Petrovic T, Logrieco A, Moretti A. 2007.
Pathogenicity and mycotoxin production by Fusarium proliferatum iso­
lated from onion and garlic in Serbia. Eur J Plant Pathol. 118:165–172.
doi:10.1007/s10658-007-9126-8.
Stępień Ł, Koczyk G, Waśkiewicz A. 2011. Genetic and phenotypic
variation of Fusarium proliferatum isolates from different host species.
J Appl Genet. 52:487. doi:10.1007/s13353-011-0059-8.
Torabi M, Ghorbany M, Salari M, Mirzaee MR. 2014. First report of
sesame wilt disease caused by Fusarium proliferatum in Iran. J Plant
Pathol. 96(S4):114.
Vujanovic V, Hamel C, Yergeau E, St-Arnaud M. 2006. Biodiversity
and biogeography of Fusarium species from northeastern North
American asparagus fields based on microbiological and molecular
approaches. Microb Ecol. 51:242–255. doi:10.1007/s00248-005-
0046-x.
Zhan R-L, Yang S-J, Ho -H-H, Liu F, Zhao Y-L, Chang J-M,
He Y-B. 2010. Mango malformation.disease in south China caused by
Fusarium proliferatum. J. Phytopathol. 158:721–725. doi:10.1111/
j.1439-0434.2010.01688.x.