The publications in this collection do
not reflect current scientific knowledge
or recommendations. These texts
represent the historic publishing
record of the Institute for Food and
Agricultural Sciences and should be
used only to trace the historic work of
the Institute and its staff. Current IFAS
research may be found on the
Electronic Data Information Source
site maintained by the Florida
Cooperative Extension Service.
Copyright 2005, Board of Trustees, University
100 Ft. Pierce ARC Research Report FTP 84-1 rebruary IYo4
ebruary 18' I
Experiments with methods of isolating Rhizoctonia solani Kuhn incitant
-* ,'\bf\Macroptilium atropurpureum foliar blight from soil.
SH. Ypema and R. M. Sonoda
reia of Rhizoctonia solani Kuhn (AG-1) obtained from diseased
SC.a tilium atropurpureum (DC) Urb. did not grow on Flowers' medium,
selective for other R. solani. Recovery of R. solani sclerotia from soil
by direct observation through a microscope was time-consuming and recovery
rate was only about 43%. A sieving-flotation-plating.technique resulted
in 50 to 90% recovery of sclerotia. A recovery rate of 50% was obtained
with a flotation system.
Rhizoctonia solani Kuhn, incitant of foliar blight of Macroptilium
atropurpureum (DC) Urb. produces sclerotia on infected or surface-colonized
leaves and stems from summer through early winter in south Florida. The
sclerotia detach easily when mycelia have dried out. Sclerotia may remain
in the soil during winter and serve as sources of inoculum in spring.
Sclerotia serve as sources of inoculum (2, 9) in bean web blight, a R.
solani-incited foliar disease. Pole beans and M. atropurpureum have some-
what similar gowth habits and are genetically close. The former, however,
is an annual crop and the latter is a perennial forage crop (3,7). Neither
the life cycle of R. solani on M. atropurpureum nor the function of sclerotia
in pathogenesis has been studied. In order to conduct life cycle studies,
suitable means of determining populations and changes in populations of this
R. solani are needed. Studies on several techniques of measuring the popula-
tion of this pathogen are reported here.
Field work was conducted in a planting of several cultivars of M.
atropurpureum, Stylosanthes spp. and Alysicarpus spp. on Oldsmar fine sand at
the Agricultural Research Center, Fort Pierce. Foliar blight incidence was
high in the summer and fall of 1983. Small brown sclerotia, 0.1 to 0.3 mm
long, of R. solani, were produced in large numbers on naturally infected
plants. Soil samples used in the following experiments were air-dried to
about 1% moisture before processing.
Flowers' medium (1) is reported to be selective for R. solani, however,
no R. solani was isolated from soil underneath infected M. atropurpureum
plants with this medium. Further tests showed that sclerotia of R. solani
Post-graduate student and Professor, University of Florida, Institute of
Food and Agricultural Sciences, Agricultural Sciences, Agricultural Research
Center, Fort Pierce, Fl 33450.
. .. .. .. -
picked from infected M. atropurpureum stems did not germinate on Flowers'
medium but germinated on other non-selective media.
Sclerotia in air-dried soil can be recognized by their shape, color
and surface structure with the aid of 10X magnification. From samples of
soil, collected from under infected M. atropurpureum, portions of approxi-
mately 0.25 gm were placed in glass petri dishes, spread, and scanned for
sclerotia at 10X. When a sclerotium was observed, its identity was con-
firmed at 50X. Sclerotia were removed from the soil with a distilled-water-
wetted transfer needle and germinated on water agar for further study. The
efficiency of the technique was tested by infesting 10 g of air dry,
sclerotia-free soil with 10 R. solani sclerotia and scanning the soil in 0.25
gram portions. In three replicate 10 g samples 7,3 and 3 sclerotia were
detected, a recovery rate of about 43%. The procedure is time-consuming as
it took 3 to 4 hours to scan 10 g of soil.
A sieving-flotation-plating technique similar to those reported pre-
viously (5,8) was developed. The procedure consists of; 1) sieving the
soil to exclude soil-particles outside the size-range of sclerotia; 2)
blending the remaining soil in a sucrose-solution to separate sand-particles
from organic matter and sclerotia; and 3) plating sclerotia embedded in
water agar blocks onto pre-poured water agar (last step: R. Ploetz, Univ.
of Florida, G'ville, pers. comm.).
Sieving--20 g samples of air dry sclerotium-free soil were infested with
20 R. solani sclerotia, collected from diseased M. atropurpureum. The
samples were wet-screened through nested 1.0 mm and 0.075 mm mesh screens
under running tap water. Because air-dry sclerotia tend to float, care
was taken to avoid splashing, which could have resulted in loss of sclerotia.
The soil remaining on the 0.075 mm mesh screen was then gently washed with
about 50 ml distilled water into a Buchner funnel and water.removed by vacuum.
The vacuum was left on for a few minutes after free moisture was removed to
partially dry the soil. The sample was processed immediately to avoid con-
fusion due to premature germination of sclerotia.
Flotation--To further separate sclerotia from heavier soil particles, sucrose
and KC1 solutions were tested as suspension media for the material remaining
on filter paper in the Buchner funnel. 'Sparkleen' detergent (Fisher Scientific
Company) was tested as an ingredient to submerge floating sclerotia. The effect
of these compounds on germination of sclerotia and initial radial growth of the
fungus was determined. About 20 sclerotia each were suspended in various
concentrations and combinations of the compounds for 20 minutes. The sclerotia
were then transferred to water agar and incubated at room temperature (20-260C).
Untreated sclerotia were transferred to water agar to serve as control. The
results of this study are shown in Table 1.
Table 1. Germination of sclerotia and relative growth of mycelia recorded
on water agar, 24 hrs. after removal from various suspending solutions.
compound conc.(% w/v) germination (%) hyphal growth (%)
none -- 100 100
sucrose 33 100 100
KC1 20 100 50
KC1 25 25 25
Sparkleen 9 75 50
Sucrose + Sparkleen 33 + 9 75 50
KC1 + Sparkleen 20 + 9 50 50
Reduction in rate of hyphal growth appeared to be temporary as growth
after 24 hrs. appeared normal (Table 1). However, to easily separate R.
solani from other organisms in the sample a rapid initial growth rate was
It was noticed that about 15 minutes after dry sclerotia were placed
on the surface of sucrose or KC1 solutions, sclerotia were wetted and
suspended. This removed the need for a detergent to wet and submerge the
sclerotia. Because of its apparent non-toxicity to R. solani, the 33%
sucrose solution was selected as the suspending solution.
The soil from the Buchner funnel (see Sieving section) was poured into
a Waring blender with approximately 500 ml 33% sucrose solution. The
suspension was allowed to stand for about.15 minutes to wet the sclerotia
and was blended gently, avoiding foaming. When heavier soil particles
separated and sank to the bottom of the blender, the sucrose solution con-
taining sclerotia and organic debris was poured into a large Buchner funnel
and vacuum applied. The filtrate was poured back into the blender and this
procedure repeated, if sclerotia and debris remained in the blender. After
most of the organic matter was transferred to the Buchner funnel and the sucrose
solution removed by vacuum, the sclerotia and organic matter on the filter
paper were washed with distilled water and dried for 2 hrs. in a forced con-
vection incubator with heating unit turned off (room temperature 20-26C).
The sclerotia and organic debris were dried for easier removal from the filter
Plating--Sclerotia and associated organic debris were scraped from the filter
paper with a transfer needle onto rectangular 100 mm x 100 mm plastic petri
dishes. Molten water agar (2%, 44 C) was added to the dishes and the agar
stirred. When solidified the agar was cut into 100 square blocks that
were transferred to plates of water agar (1.5%). The characteristic
growth pattern of R. solani was seen around the agar blocks in 1 to 4
days. It is possible that other sources of inoculum, e. g. infested
plant debris, will be detected with the above technique. Sclerotia
can also be detected in organic debris by scanning the filter paper
after drying in the forced air incubator. However, the possibility of
detecting other sources of inoculum is excluded then.
The efficiency of the sieving-flotation-plating technique was tested
with sclerotia-infested 20 g soil samples. Recoveries of 10 to 18 sclerotia
were made out of the original 20 placed in each sample.
In an attempt to take advantage of the fact that air-dry sclerotia
float on the surface of water, air-dried infested soil was placed in a
beaker placed in a funnel, and the funnel placed over a Buchner funnel
with filter paper in place and under vacuum. Tap water was slowly added
to the beaker and the water allowed to overflow through the funnel onto
the filter paper in the Buchner funnel. The soil in the beaker was stirred
gently during the process. The number of sclerotia obtained by this process
was recorded by scanning the filter paper from the Buchner funnel. The
recovery rate was about 50%. Further work with this system is desirable
because of its simplicity.
In all of the above techniques, the isolated R. solani should be com-
pared with authentic R. solani AG-1 isolates by anastomosis (4, 6).
S 1. Flowers, R. A. 1976. A selective medium for isolation of Rhizoctonia
solani from soil and plant tissue. Proc. Am. Phytopath. Soc. 1976,
2. Galindo, J. J., Abawi, G. S., Thurston, H. D. and Galvez G. 1983. Source
of inoculum and development of bean web blight in Costa Rica. Plant
3. Kretschmer, A. E., Jr. 1972. Siratro (Phaseolus atropurpureus D. C.), a
summer-growing perennial pasture legume for central and south Florida.
Fla. Agric. Exp. Stn. Circ. S-214.
4. Parmeter, J. R., Sherwood R. T. and Platt, W. D. 1969. Anastomosis grouping
among isolates of Thanatephorus cucumeris. Phytopathology 59:1270-1278.
5. Rodriguez-Kaban R., Backman, P. A. and Wiggins, E. A. 1974. Determination
of sclerotial populations of Sclerotium rolfsii in soil by a rapid
flotation sieving techique. Phytopathology 64:610-615.
6. Sherwood, R. T., 1969. Morphology and physiology in four anastomosis groups
of Thanatephorus cucumeris Phytopathology 59:1924-1929.
7. Skerman, P. J. 1977. Tropical forage legumes. FAO Plant Production and
Protection series no. 2. Edited by F. Riveros, Rome 1977. Pages 331-340.
8. Ui, T., Naiki T. and Akimoto, M. 1976. A.sieving-flotation method using
hydrogen peroxide solution for determination of sclerotial population
of Rhizoctonia solani Kuhn in soil. Ann. Phytopath. Soc. Japan 42:46-48.
9. Weber, G. F. 1939. Web blight, a disease of beans caused by Corticium
microsclerotia. Phytopathology 29:559-575.