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A selective medium for enumerating low

populations of Thielaviopsis basicola in
tobacco field soils
a a
Lawrence P. Specht & G.J. Griffin
a
Department of Plant Pathology, Physiology and Weed Science ,
Virginia Polytechnic Institute and State University , Blacksburg,
Virginia, 24061, U.S.A.
Published online: 29 Dec 2009.

To cite this article: Lawrence P. Specht & G.J. Griffin (1985) A selective medium for enumerating low
populations of Thielaviopsis basicola in tobacco field soils, Canadian Journal of Plant Pathology, 7:4,
438-441, DOI: 10.1080/07060668509501676

To link to this article: http://dx.doi.org/10.1080/07060668509501676

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 CANADIAN JOURNAL OF PLANT PATHOLOGY 7; 438-441, 1985

Note

A selective m e d i u m for enumerating low p o p u l a t i o n s o f

Thielaviopsis basicola in t o b a c c o field soils

Lawrence P. Specht and G.J. Griffin

Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University
Blacksburg, Virginia 24061, U.S.A.
Accepted for publication 1985 08 19
Downloaded by [University of Illinois at Urbana-Champaign] at 13:43 13 March 2015

A selective medium (TB-CEN) is described for the quantitative isolation of Thielaviopsis basicola from naturally infested
tobacco field soils containing low population densities. The medium contains etridiazol and nystatin as selective inhibitors of
undesired fungi, and unautoclaved carrot extract for selective enhancement. The pour-plate technique, along with incubation
at !6-22°C, yielded the greatest recovery of T. basicola on TB-CEN. The selective medium was as sensitive as carrot-disks,
and better than three other selective media, for isolating T. basicola from tobacco field soils.
Specht, L.P.. and Griffin, G.J. 1985. A selective medium for enumerating low populations of Thielaviopsis basicola in tobacco field
soils. Can. J. Plant Pathol. 7: 438-441.

On décrit un milieu sélectif (TB-CEN) pour l'isolation quantitative de Thielaviopsis a partir des sols des champs de tabac
naturellement infestés de populations de faibles densités. Le milieu contient de l'étrédiazol et de la nystatine comme
inhibiteurs sélectifs de champignons indésirables et de Textrait de carotte non autoclave comme stimulant sélectif. La
technique de la plaque de Petri incubésa I6-22°C a permis la plus grande recuperation de T. basicola sur TB-CEN. Le milieu
sélectif a été aussi sensible que les disques de carotte et meilleur que trois autres milieux sélectifs pour l'isolation de T. basicola
des sols des champs de tabac.

Thielaviopsis basicola (Berk. & Br.) Ferraris
(= Chalara elegans Nag Raj and Kendrick) is a
widespread plant pathogen that causes black root
rot on a variety of crops. Present methods for the
quantitative assay of this fungus in Virginia
tobacco field soils are not satisfactory. Various
modifications of Yarwood's carrot-disk procedure
(1946), such as the most probable number method
(Tsao & Canetta 1964), can provide quantitative
population data. However these procedures are
time consuming, do not enumerate propagule
numbers as discrete colonies, and require a large
number of replicates for statistical accuracy. The
most desirable method for enumerating T. basicola
is the dilution-plate technique. Several selective
media have been reported, including RB-M2
medium (Tsao 1964), V D Y A - P C N B medium
(Papavizas 1964), and T B M - C and TM V-V8 media
(Maduewesi et al. 1976). In preliminary trials, none
of these media satisfactorily inhibited undesired
fungi at low soil dilutions, particularly at 10~' dilu-
tion. Consequently a new selective medium was
developed for the purpose of enumerating T. basic-
ola present in tobacco field soils.
The medium is called T. basico/a-carrot-
etridiazol-nystatin (TB-CEN) medium. The per L
composition was 80 m L o f 5 0 % ( w / v ) unautoclaved
carrot extract, 400 mg etridiazol (5-ethoxy-3-
trichloromethyl-l,2,4-thiadiazole, added as 1.14 g
Terrazole 35WP©, Uniroyal Corp.), 250 000 units
nystatin, 500 mg streptomycin sulfate, 30 mgchlor-
tetracycline hydrochloride, I g C a C 0 3 , and 15 g
agar. The carrot extract was prepared by blending
(Waring Blendor at high speed for 2 min) 100 g of
fresh, peeled, raw carrots with 100 mL distilled
water. After blending, the solid material was
removed by straining through several layers of
cheesecloth. All components were added to molten
water agar immediately prior to use, with the anti-
biotics being added as solutions. The pH of the
medium was adjusted to 5.3 with 1 N H 2 S 0 4 .
The following pour-plate procedure was used for
assaying T. basicola. One-mL aliquots of soil sus-
pensions were pipetted into empty glass petri plates.
Twenty-seven to 30 mL of molten (48° C) TB-CEN
medium was then poured into each plate and the
agar agitated to distribute the soil particles. The
medium was poured while swirling the flask to keep
the fungicide uniformly suspended. Except for an
incubation-temperature study, plates were kept at
room temperature (20-22° C) for 14 days prior to
counting colonies.
Recovery of T. basicola from 10 naturally
infested tobacco field soils using TB-CEN was con-
sistently better than with T B M - C , T B M - V 8 , and
V D Y A - P C N B media (Table I). Figure I shows the
appearance of colonies of T. basicola and other
fungi that developed on the soil dilution plates. The

438
 SPECHT, GRIFFIN: TOBACCO/THIELAVIOPSIS 439
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Figure 1. Soil dilution plates prepared from a tobacco field soil naturally infested with Thielaviopsis basicola. The pour-plate
technique was used in combination with 10'1 soil dilutions for A) TB-CEN, B) TBM-C, C) TBM-V8 and D) VDY A-PCNB media.
Arrows indicate typical colonies of T. basicola. No colonies of T. basicola were recovered on VDYA-PCNB medium.

TB-CEN medium gave recovery rates similar to
those achieved using a modification (Rittenhouse
1982) of Yarwood's carrot-disk procedure (Table
2). The recovery of T. basicola, using various assay
techniques, from soil collected from around the
roots of tobacco plants with black root rot is shown
in Table 3. The recovery of T. basicola was signifi-
cantly greatest when glass petri plates were used.
The recovery of other fungi also was more of a
problem on TB-CEN medium in plastic petri plates
(which may adsorb the etridiazol) than in glass petri
plates. In this study (Table 3), using soil with a high
population density of T. basicola, the pour-plate
technique was not significantly better than the
spread-plate technique when TB-CEN was
employed. However in another study with soil hav-
ing a low population density of T. basicola and
using a 10"' soil dilution, the pour-plate technique
was superior to the spread-plate technique.
The percent germination of endoconidia and
 440 CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 7, 1985

Table 1 . Relative recovery of Thielaviopsis basicola obtained Table 3. Relative recovery of Thielaviopsis basicola from a nat-
with four selective media from naturally infested tobacco field urally infested tobacco field soil using 10assay techniques
soils usi ng 10 soil dilutions
Propagules per g soil3
No. propagules per g soil3 Pour-plate Spread-plate
Soil code TB-CEN TBM-C TBM-V8 VDYA-PCNB Mediumb technique technique
I 13 Ab 0B 0B 0B TB-CEN (G) 2I10A C 1900 A
2 20 A 0B 0B 0B TB-CEN (P)*d 1410 B 830 C
3 30 A 0B 0B 0B TBM-C (G) 1500 B 1460 B
4 74 A 21 B 0C OC TBM-V8{G)* I260B 840 C
5 92 A 40 B 13 C 0C VDYA-PCNB (G)* 410 C 30 D
6 94 A 50 B 0C OC "Soil sample assayed, us ing a I0"2 dilution, was collected from
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7 120 A 7B 8B 2B around the root system of tobacco plant roots colonized by T.

8 167 A 27 B 2C 0C basicola.
9 214 A 132B 13C OC b
Glass (G) or plastic (P) petri plates tested.
10 422 A 340 B 73 C 0D c
Values followed by the «same letter are not significantly differ-
"Selective media used in combination with pour-plate tech- ent (5% level) according to the Duncan's Multiple Range test.
d
nique. Ten plates of each medium were used per soil sample. Asterisk indicates the pour-plate technique is significantly dif-
TB-CEN plates were incubated for 14daysand the other three ferent (5% level) from the spread-plate technique, as deter-
media for 6 days. mined by a t-test.
b
Values followed by the same letter are not significantly differ-
ent (5% level) accord ing to the Duncan's multiple range test.
Statistical comparisons were made horizontally among media,
and not vertically am ong soils. Fewest undesired fungi were present at the lowest
temperature, but a long incubation period (21 days)
was required for complete colony development.
Table 2. Relative recovery of Thielaviopsis basicola from natu-
rally infested tobacco field soils using TB-CEN medium and
Other tests indicated that the recovery of T. basic-
carrot-disks ola was the same whether plates were incubated
under dark or light conditions. The new medium
Propagules per g soil was also used for isolating T. basicola from colon-
Soil cod e TB-CEN ab Carrot diskc ized plant roots. The best result were obtained when
tl 0 8.9 root segments were washed in running tap water, or
12 0.5± ld 0 in a distilled-water blank on a wrist-action shaker,
13 4.3± 2 0 and also when a thin layer of TB-CEN medium was
14 8.4± 3 9.1 poured over the root segments after placing them
15 12 ± 5 0
16 26 ± 5 42 on the surface of the medium.
17 76 ± 14 97 The medium described here used nystatin and
18 101 ± 18 82 etridiazol for selective inhibition. Etridiazol has a
19 166 ± 17 226 wide spectrum of antifungal activity and is espe-
"Pour-plate technique iused with 10"' soil dilutions. cially effective against Oomycetes. This fungicide
b
Twenty plates per soil sample prepared. controlled many soil fusaria that were not ade-
c
Twenty carrot disks per soil sample, and data were corrected
for multiple colonizat ion (Vanderplank 1963). quately inhibited on the other media. Unautoclaved
d
95% confidence interval. extract from carrot (Daucus carota L. cv. Sativa)
was used to selectively enhance the growth of T.
basicola. Preliminary tests indicated that the stimu-
latory property of carrot extract was partially des-
chlamydospores of eleven isolates of T. basicola on troyed by autoclaving. The mechanism which oper-
TB-CEN was 90-97% and 92-100%, respectively. ates in carrot tissue and carrot extrct for the
When the carrot source was examined there were selective isolation of T. basicola is not known; how-
no significant differences in the performance of the ever the selective property in carrot also occurs to a
medium among the seven carrot sources tested; lesser extent in other species of Umbelliferae (Mcll-
however with two of the sources the colonies of T. veen & Edgington 1972).
basicola were less pigmented and more difficult to
count. The temperature at which TB-CEN medium This research was supported by the Virginia Bright Flue-Cured
plates were incubated had a significant effect on the Tobacco Commission and the R.J. Reynolds Tobacco Com-
recovery of T. basicola. Recovery of T. basicola pany. We thank J.E. DeVay, J.L. Lockwood, and G.C. Papavi-
zas for some of the isolates of T. basicola used in this study, and
from naturally infested soil was reduced by 50% the Uniroyal Corp. for the samples of Terrazole 35WP© pro-
when the plates were incubated at 27°C, as com- vided. We thank J. Hendrix and W. Wills for reviewing the
pared to room temperature (20-22°C) or 16°C. manuscript.

Maduewesi, J.N.C., B. Sneh, and J.L. Lockwood. 1976.
Improved selective media for estimating populations of Thie-
iaviopsis basicola in soil on dilution plates. Phytopathology
66:526-530.
Mcllveen, W.D.,and L.V. Edgington. 1972. Isolation of Thiela-
viopsis basicola from soil with umbelliferous root tissue as
baits. Can. J. Bot. 50:1363-1366.
Papavizas, G.C, 1964. New medium for the isolation of Thiela-
viopsis basicola on dilution plates from soil and rhizosphere.
Phytopathology 54:1475-1481.
Rittenhouse, C M . 1982. Inoculum pattern and relationship
between incidence of black root rot of tobacco and inoculum
density of Thielaviopsis basicola in field soil. MS Thesis,
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SPECHT, GRIFFIN: TQBACCO/THIELAVIOPSIS 441
Virginia Polytechnic Institute and State University, Blacks-
burg. 82 pp.
Tsao, P.H. I964. Effect of certain fungal isolation agar media on
Thielaviopsis basicola and on its recovery in soil dilution
plates. Phytopathology 54:548-555.
Tsao, P.H., and A.C. Canetta. 1964. Comparative study of
quantitative methods for estimating the population of Thiela-
viopsis basicola in soil. Phytopathology 54:633-635.
Vanderplank, J. 1963. Plant disease: Epidemics and control.
Academic Press, New York, NY 349 pp.
Yarwood, C.E. 1946. Isolation of Thielaviopsis basicola from
soil by means of carrot disks. Mycologia 38:346-348.