Australasian Plant Pathology (2021) 50:319–322

https://doi.org/10.1007/s13313-021-00775-3

RESEARCH NOTE

A simple carrot agar medium for isolation of black root rot pathogen
of cotton seedlings
Duy Phu Le1

Received: 23 September 2020 / Accepted: 25 January 2021 / Published online: 15 February 2021
© Crown 2021

Abstract
Black root rot (BRR) is a major seedling disease in cotton in Australia. BRR is caused by a soilborne fungus Thielaviopsis
basicola, recently re-described as Berkelyomyces spp., that was reported for the first time in 1990 in northern New South
Wales (NSW), Australia. The disease is now prevalent across NSW. Since the first detection, much research has been focused
exclusively on management; however, little has been investigated to understand the BRR pathogen population. Isolation and
collection of pure fungal cultures are essential for investigation of the pathogen diversity and pathogenicity. However, isola-
tion of T. basicola is recalcitrant. In this study, T. basicola were successfully recovered from BRR diseased cotton seedlings
in the past three seasons by using a simple 5% carrot agar amended with 100 ppm streptomycin. T. basicola was recovered
within three days with the percentage of recovery ranging from 55–76% during the first isolation attempt. This carrot medium
provided a simple and vigorous means for isolation of T. basicola.

Keywords Gossypium hirsutum · Seedling disease · Selective media · TB-CEN · Carrot discs · Water agar

Cotton (Gossypium hirsutum L.) is a major summer crop
in regional New South Wales (NSW) and Queensland in
Australia, generating an average revenue of approximately
AUD 1.9 billion in exports (CRDC 2018). Disease is a
major constraint for Australian cotton production (Kirkby
et al. 2013; Le and Gregson 2019; Le et al. 2020; Nehl et al.
2004). Black root rot (BRR) is one of the main seedling
diseases of cotton in NSW (Kirkby et al. 2013; Nehl et al.
2004). The disease was first reported in Australia in 1990
by Allen in northern NSW, then spread across the industry
(Kirkby et al. 2013; Nehl et al. 2004). BBR is characterized
by dark brown to black necrotic lesions on the main and
lateral roots, which result in stunted and less vigorous
seedlings, and delayed flowering and maturity (Nehl et al.
2004). BRR is presumptively not associated with seedling
mortality directly; however, Mauk and Hine (1988) found
that death of cotton seedlings in cotton fields in Arizona
during the 1986 and 1988 seasons was associated solely
to severe BRR. In Australian cotton systems, Nehl et al.
(2004) found that yield reduction was negatively correlated
* Duy Phu Le
duy.le@dpi.nsw.gov.au; lephuduy08@gmail.com
1
New South Wales Department of Primary Industries,
2390 Narrabri, Australia
with BRR incidence, but there was no general relationship
between BRR incidence and seedling mortality. A recent
survey of consultants by Otto (2020) revealed that 6% of a
total 56 respondents experienced a reduction in revenue of
more than AUD 300 per hectare which was equivalent to an
approximately 10% yield reduction, with BRR cited as the
primary cause.
The disease is caused by a hemibiotroph, Thielaviopsis
basicola (syn. Chalara elegans) (Nehl et al. 2004; Allen
1990; Holtz and Weinhold 1994). Recently, T. basicola has
been reclassified in a new genus, namely Berkeleyomyces
gen. nov. (Nel et al. 2018). The genus accommodated two
closely related species, including B. basicola and B. rouxiae
which were only delineated by multiple sequences (Nel et al.
2018). Additionally, species status of T. basicola in Australia
has not been resolved. Therefore, unless otherwise stated,
the old name T. basicola was retained to refer to the BRR
pathogen of cotton in order to avoid incorrect citation of the
fungal name that was identified before Nel et al. (2018). T.
basicola is soilborne and characterised by production of two
spore types, including thin wall endoconidia and dark thick
wall chlamydospores (Nel et al. 2019; Paulin-Mahady et al.
2002). T. basicola has a wide host range of more than 170
host species (Rothrock 1999; Nel et al. 2019). Although T.

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basicola isolates originated from different hosts may show a
certain level of host specificity or preference, pathogenicity
of a number of isolates was not limited to its original host
crops (Allen 1990; Mondal et al. 2004; Pereg 2013).
T. basicola, including B. basicola and B. rouxiae is
relatively slow growing. At the optimal growth temperature
of 25 °C, growth diameter was recorded at 44–47 mm
per 10 days (Nel et al. 2018). Therefore, the recovery of
T. basicola from diseased plant materials was recalcitrant
(Allen 1990; Mondal et al. 2004; O’Brien and Davis 1994).
Specht and Griffin (1985) developed a selective medium
called T. basicola-carrot-etridiazol-nystatin (TB-CEN)
for quantification of T. basicola in soil samples. TB-CEN
was also commonly used for isolation of T. basicola from
diseased plant materials (McGovern and Seijo 1999;
Mondal et al. 2004; Monfort et al. 2010). The carrot disc
technique was believed to be a cheap, simple and highly
consistent in manipulating the sporulation of T. basicola,
from which spore masses were transferred onto a desired
isolating medium such as potato dextrose agar (PDA)
(Allen 1990; O’Brien and Davis 1994). Recently, Nakane
et al. (2019) successfully recovered B. rouxiae (within
T. basicola) from diseased lettuce root using only 1.5%
water agar (WA) amended with 300 ppm chloramphenicol,
300 ppm streptomycin (Table 1). Both carrot disc and water
agar isolation protocols required an additional microscopic
examination for presence of characteristic pigmented
chlamydospores before subculturing (Allen 1990; Usami T.
per. comm.). Additionally, these isolation protocol required
an incubation of 7–14 days (Table 1). In this study, I report
a simple and vigorous isolation protocol for T. basicola on
cotton seedlings using 5% carrot agar medium amended with
100 ppm streptomycin, which was modified from a carrot
juice agar reported by Baxter and Fagan (1974).
Table 1  A brief comparison of commonly used protocols for isolation of T. basicola from diseased plant materials with our carrot agar protocol
Protocols used TB-CEN
Medium constituents As per Specht and Griffin, 1985 2
Running tap-water wash Not applicable
Surface decontaminants 10% sodium hypochlorite, sterile
water
Incubation time 14 days
Incubation temperature 21 °C
References McGovern and Seijo 1999; Monfort
et al. 2010
1
Water agar was used for isolation of B. rouxiae (Nakane et al. 2019)
2
One litre of TB-CEN medium contains: 80 mL of 50% unautoclaved carrot extract, 400 mg etridiazol, 250,000 units nystatin, 500 mg strepto-
mycin sulphate, 30 mg chlortetracycline hydrochloride, 1 g C
­ aCO3 and 15 g agar (Specht and Griffin 1985)
13
D. P. Le
To prepare one litre of 5% carrot agar (CA), fresh
marketed carrot was purchased from a local supermarket,
peeled, quickly washed under running tap-water and then
chopped into small chunks. Fifty grams of the chopped
carrot were blended with 200 mL of mili-Q water (obtained
from a Barnstead™ Pacific TII water purification system) at
high speed for two minutes. The puree was drained through
a 335-µm-meshed sieve. The filtrate amended with 20 g
agar (Difco) were made up to one litre and then autoclaved
at 121 °C for 20 min. Then a 55 °C molten medium was
thoroughly mixed with 100 ppm streptomycin and poured
into gamma sterilised 90-mm-dia Petri plates (TechnoPlas)
under aseptic conditions.
Suspected BBR diseased cotton seedlings (Fig. 1)
sampled during the early season disease survey between
2017 to 2020 were subjected to isolation using the above
streptomycin amended CA medium (sCA). The isolation
was initiated with surface decontamination using 70%
ethanol (v/v). Below ground parts of the diseased seedlings
were sprayed until run off with 70% ethanol and quickly
blotted dry with paper towels. Under aseptic conditions,
small segments (3–5 mm) of diseased lateral/tap roots
were excised and embedded into the sCA plates. There
were seven segments per plate and at least two plates per
sample. The plates were sealed with parafilm and incubated
at 25 °C in darkness for at least three days. Characteristic
olive-green colonies (Fig. 1) of putative T. basicola growing
out from the diseased sections were subcultured on half
strength PDA (Difco) amended with 100 ppm streptomycin
and single spored where required. Pure cultures growing
on half strength PDA were excised and submerged under
sterile water in seal-tight vials for long term storage in the
Pathology Laboratory, Australian Cotton Research Institute,
Narrabri, NSW Australia.
Carrot disc Water agar 1 Carrot agar
Fresh carrot discs 1.5% agar, 300 ppm chlorampheni- 2% agar, 5% carrot,
col, 300 ppm streptomycin 100 ppm strepto-
mycin
Not applicable 1h Not applicable
Sterile water 70% ethanol, 10% sodium hypochlo- 70% ethanol
rite, sterile water
14 days 7–14 days 3 days
20–25 °C 25 °C 25 °C
Allen 1990; Nakane et al. 2019; Usami T. per. This study
O’Brien and comm
Davis 1994
A simple carrot agar medium for isolation of black root rot pathogen of cotton seedlings 321

Fig. 1  Field cotton seedlings

exhibiting typical black root rot
on tap and lateral roots, severe
infection caused girdling around
the collar region (red arrows)
(a); typical olive-green colonies
of T. basicola growing out of
the diseased tissue embedded in
sCA at 3 days after incubation
at 25 °C (b)

A total of 92 BRR seedling samples were processed
for the T. basicola isolation in the past three seasons. In
the first attempt, T. basicola was recovered out of 22, 16
and 19 samples in the three seasons from 2017 to 2020,
respectively. The recovery percentage of T. basicola on sCA
ranged from 55 to 76%; and the recovery frequency was
between 7 to 85.7% (Table 2). sCA was simple to prepare
and efficient enough for T. basicola isolation. T. basicola
formed olive-green colonies out of diseased tissue on
sCA. This was distinctive from other fast-growing species,
including Fusarium spp. and Rhizoctonia spp., so did not
require an additional microscopic assessment to confirm the
presence of T. basicola chlamydospores before subculturing.
The recovered isolates were further subjected to sequencing
analyses of the internal transcribed spacer and the RNA
polymerase II second largest subunit for confirmation of
the identity (unpublished data). Additionally, sCA was
sufficient to control bacterial contaminations, which allowed
Table 2  A summary of total number of samples processed, recovery
percentage and frequency of BRR pathogen of cotton seedlings using
carrot agar over the past three seasons in NSW
Seasons 2017–2018 2018–2019 2019–2020
Total samples 1
29 29 34
Recovery percentage 2 75.9 55.2 55.9
Recovery frequency (%) 3 7–78.5 7–85.7 14–57.1
1
Cotton seedlings exhibited characteristic symptoms of BRR were
sampled during the early season disease surveys undertaking between
4–6 weeks after planting
2
Recovery percentage indicated the successful recovery of BRR
pathogen from the samples during the first isolation attempt
3
Recovery frequency (%) was calculated based on the number BRR
isolates recovered from the 14 root segments plated out on the sCA
medium
for the adoption of a less aggressive surface decontamination
by spraying with only 70% ethanol. Subsequently, T.
basicola was recovered within three days after incubation
in comparison to 14 days using other protocols (Table 1).
In summary, the sCA currently being used in our Narrabri
laboratory is simple and vigorous for isolation and collection
of T. basicola from BRR diseased cotton seedlings.
Acknowledgements This research was supported through funding
from the Cotton Research and Development Corporation and NSW
Department of Primary Industries (projects DAN2101 and DAQ2002).
I greatly thank CottonInfo team for sampling assistance; and Aphrika
Gregson from NSW DPI Narrabri for technical assistance. I specially
thank Dr Bernie Dominiak from NSW DPI Orange for critically
reviewing and commenting for the improvement of the manuscript.
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