Annals of Botany 78 : 653–657, 1996
Production of Microconidia by Cercospora henningsii Allesch, Cause of Brown
Leaf Spot of Cassava (Manihot esculenta Crantz) and Tree Cassava (Manihot
glaziovii Muell.-Arg.)
E. N. A Y E SU-O F F E I and C. A N T WI-B O A S I A K O
Department of Biological Sciences, UniŠersity of Science and Technology, Kumasi, Ghana
Received : 28 June 1995
Accepted : 31 May 1996
Sporulation of Cercospora henningsii Allesch has been examined under various relative humidities, and in the presence
of free water in lesions on leaves of cassava and tree cassava. Mature conidia of the fungus on both cassava and tree
cassava do not germinate in lesions but accumulate, and under the optimum conditions of 25–32 °C and in the
presence of free water, they bud and fragment into numerous microconidia. Microconidia are cylindrical, mostly onecelled, and measure 7±5–17±5¬3±7–7±5 µm. Production of microconidia significantly decreases as relative humidity
decreases. Microconidia readily germinate by means of a germ tube at 100 % relative humidity on both surfaces of
host leaf and on glass slides. Some germ tubes form appressoria and symptoms appear on cassava leaves inoculated
with microconidia. The results are discussed in relation to possible modes of dispersal of the spores and control of
the disease.
# 1996 Annals of Botany Company
Key words : Free water, sporulation, budding, microconidia.
INTRODUCTION
Cassava (Manihot esculenta Crantz) is cultivated in many
tropical areas of the world, notably in tropical Africa, Latin
America and Asia. An estimated 300–500 million people in
the areas mentioned above, rely on cassava as a major
source of carbohydrate (Bellotti and Van Schoonhoven,
1978). Both the tubers and leaves are widely consumed.
It has been reported that brown leaf spot of cassava is
found wherever the crop is grown (Jameson, 1970 ; Lozano
and Booth, 1974). It is the most important fungal disease of
cassava and, in Ghana, it seems almost all cassava plants
are affected by the disease. Severe incidences of the disease
have been noted, particularly under wet conditions, in a
number of African countries. The disease can cause great
loss in leaf and tuber yield (Terry and Oyekan, 1976).
The causal fungus has been identified by a number of
workers including Leather in Ghana (Leather, 1959) as
Cercospora henningsii Allesch, an obligate parasite in the
field. Leather (1959) also reported the occurrence of the
disease on tree cassava (Manihot glazioŠii Muell.-Arg.) in
Ghana.
The fully formed lesions, on both cassava and tree
cassava, are large, necrotic spots, circular (5–12 mm
diameter) or irregular in shape and dark brown on both
surfaces of the leaf, sometimes with a distinct dark border
on the abaxial surface. The conidia of the fungus are
described in the literature as filiform, 3–10 septate and
30–85¬5–7 µm in size.
Observations by the senior author on sporulation of the
fungus on naturally infected leaves of cassava and tree
cassava, revealed that in addition to conidia of the fungus
described in the literature, there was always another type of
0305-7364}96}110653­05 $25.00}0
spore present in sporulating lesions. Since no published
work is available concerning these spores, investigations
were initiated to provide some information on their origin
and possible role in the infection process.
MATERIALS AND METHODS
The varieties of cassava (Ankra) and tree cassava used in the
experiments were planted on an experimental plot at the
Department of Biological Sciences, University of Science
and Technology, Kumasi, Ghana. They were naturally
infected and were the source of lesions for the investigations.
For each experiment, except where otherwise stated,
lesions on infected leaves of both cassava and tree cassava
were thoroughly washed with water using a soft wad of
cotton wool. This treatment removed spores from the
surfaces of the lesions. The lesions were cut out from the
leaves and immersed in a solution of equal parts of 95 %
alcohol and 0±001 % HgCl solution for 2 min in order to
#
sterilize their surfaces. Next they were thoroughly washed in
two changes of sterile distilled water. The wet lesions were
then placed in sterile Petri dishes lined with wet filter papers
and incubated in the dark at room temperature (25–32 °C)
to sporulate.
Studies on leaf-borne spores
To study the types of spores formed during sporulation,
the wet sterilized lesions were incubated for 48 h. Some wet
unsterilized lesions were similarly incubated. At the end of
the incubation period, clean slides were brought into contact
with sporulating lesions so that some spores (spore print)
adhered to them. The spores were stained with cotton blue
# 1996 Annals of Botany Company
654 Ayesu-Offei and Antwi-Boasiako—Production of Microconidia by Cercospora henningsii Allesch
in lactophenol. Camera lucida drawings were made of the
spore types observed. Lengths and widths of at least 200
spores of each kind were measured. A notable observation
in the present work on the conidia of the fungus already
described in the literature, was their ability to bud and also
to fragment into smaller portions. Camera lucida drawings
were therefore made to illustrate these processes.
Effect of incubation period on frequency of types of spores
formed in lesions
Five wet surface-sterilized sporulating lesions (from
cassava) were randomly taken from Petri dishes in an
incubator at 24 h intervals beginning from 24 h after
incubation. Each group of five lesions was shaken in a test
tube containing 1 ml distilled water to make a spore
suspension. 10 µl of spore suspension was placed on a slide.
After the water had evaporated, 20 µl of cotton blue in
lactophenol was dropped onto the spore print. A cover slip
(16 mm diameter) was placed on the stained spores and the
number of each type of spore in ten microscope fields
(¬100) were counted. The experiment was repeated four
times. In each experiment, unsterilized but thoroughly
washed lesions were used for a second test.
Effect of ambient humidity on sporulation and frequency of
each spore type
Wet surface-sterilized lesions were dried under a fan.
Sulphuric acid solutions were prepared and placed in plastic
chambers (12¬10¬8 cm) with tightly fitting lids to maintain
relative humidities of 40, 60, 80, 85, 90 and 95 %, respectively (Solomon, 1952). Each chamber held 10 ml of the
appropriate solution to give the desired humidity. Ten
millilitres of sterile distilled water was used where a relative
humidity of 100 % was required. Ten dry lesions were put
into each of a number of watch glasses. A watch glass and
its contents was supported at the bottom of one of the
humidity chambers by a solid watch glass. Each was
incubated at 25–32 °C for 48 h.
At the end of the incubation period in each experiment,
1 ml of distilled water was added to five lesions at each
humidity level and spore suspensions were prepared as
previously described. Slides were made for each humidity
level and spores were counted as already described. The
experiment was repeated four times.
generally contained mainly microconidia. Hence, to inoculate with microconidia, spores were transferred from
lesions which had been incubated for 6 d into drops of
distilled water on slides. Each spore suspension was
thoroughly examined under the microscope and those
which contained only microconidia were used for the
inoculation. Inoculated plants were incubated in humid
chambers in a glass house (25–32 °C). After 48 h, some
inoculated areas were cut out and cleared in a mixture of
equal parts (v}v) of glacial acetic acid and absolute alcohol,
stained with modified acid–schiff reagent (Ayesu-Offei and
Clare, 1970) and mounted in dilute glycerine for observation
under the microscope. The cassava plants were removed
from the humid chambers after 48 h incubation and the
remaining inoculated spots were observed for a period of
30 d for symptoms.
RESULTS
Studies on leaf-borne spores
Surface sterilized and unsterilized lesions from both cassava
and tree cassava sporulated abundantly. Each lesion from
both cassava and tree cassava contained two spore-forms.
The larger of the two forms, termed macroconidia in this
paper, has been described for C. henningsii obtained from
cassava. The results obtained in the present study are
presented in Fig. 1 (A, B, C) and Table 1 and are in general
agreement with those already published.
Of much interest in the present work were smaller-sized
spores which were previously unknown. They are referred
to as microconidia in this paper. They were cylindrical and
pa
pa
D
E
B
Germination of microconidia and infection tests
In studies on glass slides, spores from sporulating lesions
were deposited on slides. A drop of sterile distilled water
was put on each spore print and the slides were placed in
humidity chambers at 100 % relative humidity. They were
incubated in the dark (25–32 °C) for 48 h. The incubated
spores were air dried and mounted in cotton blue in
lactophenol for microscopic examination. In studies on host
leaf surfaces, microconidia were deposited on marked areas
on both abaxial and adaxial surfaces of leaves of cassava
plants growing in pots. Lesions incubated for approx. 6 d
F
C
A
10
10 µm
µm
G
pa
F. 1. Camera lucida drawings of spores of C. henningsii. A, B, C,
Macroconidia ; D, E, F, G, microconidia ; pa, papilla.
Ayesu-Offei and Antwi-Boasiako—Production of Microconidia by Cercospora henningsii Allesch 655
T     1. Frequency distribution of lengths and widths of spores of C. henningsii formed in lesions on cassaŠa
Macroconidia
Length of
spore (µm)
Number of
spores
counted
20–39
40–59
60–79
80–99
100–119
180
201
85
33
1
40±5³0±81*
Microconidia
Width of
spore (µm)
Number of
spores
counted
5–5±9
6–6±9
7–7±9
475
18
7
5±5³0±01*
Length of
spore (µm)
7–8±9
9–10±9
11–12±9
13–14±9
15–16±9
17–18±9
11±2³0±09*
Number of
spores
counted
57
210
131
80
13
9
Width of
spore (µm)
Number of
spores
counted
3–3±9
4–4±9
5–5±9
6–6±9
7–7±9
7
10
380
82
21
5±6³0±03*
* Mean³standard error.
separates from the rest of the spores by abstriction (Fig.
2 C). The protoplast is then set free to develop into an
independent microconidium. The remaining cells of the
macroconidium then moved out of the disintegrating cell
wall to develop into microconidia.
A similar constriction may divide any cell of the
macroconidium into microconidia followed by the release of
the remaining cells. Any of the cells of the macroconidium
may also put out a lateral bud, which is then liberated to
develop into a microconidium. The cell wall of any
macroconidium may also disintegrate to release the cells to
develop into microconidia (Fig. 2 A, B).
10 µm
Effect of incubation period on the frequency of spores
formed in lesions
A
B
C
D
F. 2. Camera lucida drawings illustrating budding and fragmentation
of macroconidia of C. henningsii derived from cassava. D, Macroconidium with a globular bud formed from one of the terminal cells ; C,
macroconidium after the bud had separated by abstriction ; A, B,
disintegration of cell walls to release cells of the macroconidium.
mostly one-celled. A few were medianly septate. They
measured 7±5–17±5¬3±7–7±5 µm (Fig. 1 D–G, Table 1).
Unlike the macroconidia, they had no papillae and had
varying numbers of prominent vacuoles in their cytoplasm.
Formation of microconidia
Our observations indicated that the microconidia were
formed by means of budding and fragmentation of the
macroconidia. Commonly, a constriction occurs at one end
of the macroconidium. The terminal cell of that end of the
spore then becomes completely surrounded by the cell wall
to form a small globular bud (Fig. 2 D). The bud then
In all four experiments, there were more macroconidia
than microconidia in the wet sporulating lesions at the end
of 24 h of incubation. But the proportion of microconidia
increased with increase in duration of the incubation period.
The increase was already apparent where lesions had been
incubated for 48 h. After 144 h of incubation, almost all the
spores in the lesions were microconidia. The mean number
of spores and standard error of each group of four
experiments are given in Table 2.
Effect of ambient humidity on sporulation
The total number of microconidia produced after 48 h of
incubation at 100 % relative humidity was significantly
higher than the total number of macroconidia produced
under the same conditions (P ! 0±001). The mean of the
four experiments and the standard error at each humidity
level are presented in Table 3. Production of both types of
spores decreased as the humidity decreased, but the greater
magnitude of effect of reduced humidity was on the
microconidia whose production had almost ceased at 40 %
relative humidity (Table 3).
Germination of spores and infection tests
The microconidia germinated on both surfaces of the host
leaf and on glass slides at 100 % relative humidity (Fig. 3).
Appressoria were formed at the apices of germ tubes.
656 Ayesu-Offei and Antwi-Boasiako—Production of Microconidia by Cercospora henningsii Allesch
T     2. The effect of incubation period on sporulation and frequency of spore-types of C. henningsii in sterilized and
unsterilized lesions from cassaŠa incubated at 25–32 °C
Mean number of spores³s.e.
Length of
incubation
period (h)
Sterilized lesions
Macroconidia
Microconidia
Macroconidia
Microconidia
24
48
72
96
120
144
249±0³47±6
242±8³89±4
189±5³82±6
147±0³58±9
95±0³29±4
54±0³14±9
88±3³21±3
966±5³218±9
992±3³298±7
1867±5³536±5
2435±8³341±8
3493±8³1087±7
651±0³33±8
244±5³21±3
147±5³29±2
116±8³18±1
91±0³15±9
41±0³11±5
42±5³7±1
847±5³447±1
1783±8³97±0
2050±5³655±0
3089³755±3
3500±0³703±2
T     3. Effect of ambient humidity on sporulation and
frequency of spores of C. henningsii in sterilized lesions from
cassaŠa, 48 h after incubation at 25–32 °C
Mean number of spores³s.e.
% Relative
humidity
Macroconidia
Microconidia
100
95
90
85
80
60
40
270±5³56±3
279±8³30±8
195±0³23±1
161±3³27±2
140±8³25±5
149±8³11±3
109±3³13±8
639±5³96±3
351±0³35±2
76±0³9±6
53±0³12±9
12±8³4±1
11±8³1±5
6±3³0±63
gt
ap
10 µm
F. 3. Camera lucida drawings of germinating microconidia of C.
henningsii. gt, Germ tube ; ap, appressorium.
Water-soaked areas developed at about 30–40 % of the
points of inoculation with microconidia on the abaxial
surfaces of the leaves within 9–18 d after inoculation. Fully
formed lesions appeared 20–30 d after inoculation. Infection
rarely occurred on the adaxial surface of leaves.
Unsterilized lesions
DISCUSSION
The work reported in this paper has shown that Cercospora
henningsii Allesch produces numerous microconidia. The
microconidia germinated by means of germ tubes, some of
which formed appressoria. Appressoria are formed by
germinating spores of some types of parasitic fungi and are
often sites at which infection hyphae are formed. Inoculation
of cassava, by the authors, with spores from lesions on
cassava, readily infected cassava plants. Macroconidia and
microconidia generally occurred together in lesions but by
incubating lesions for six or more days, it was possible to
select spore prints which contained only microconidia and
to demonstrate that they alone could cause infection. The
authors are unaware of any previous knowledge of the production of microconidia by C. henningsii. These investigations have therefore demonstrated that the fungus produces
a larger number of inocula than was previously thought.
The observation from the present study is that many of
the microconidia budded off from cells of the macroconidia
and are therefore blastospores (cf. Talbot, 1971). But a
significant number of them were formed by fragmentation
of the macroconidia and therefore these could not be
described as blastospores.
The studies showed that the longer the incubation period
of wet lesions, the greater the number of ungerminated
spores formed in the lesions, most of them microconidia.
This shows that mature spores failed to germinate while still
in lesions. It has been suggested that in some fungi autoinhibitors produced by spores prevent germination in
sporulating lesions (Pritchard and Bell, 1967 ; Ayres and
Owen, 1970). In the presence of free water, the macroconidia
of C. henningsii in lesions, therefore, bud and fragment into
numerous microconidia which could then be dispersed by
rain drops. Unpublished observations by the authors
strongly suggest that rain-splash dispersal of the spores is
more important in dispersing secondary inoculum within a
farm or plantation than other modes of dissemination.
Ayesu-Offei and Carter (1971) made similar observations on
leaf scald of barley. The biological advantage of the above
observations with regard to water is that numerous
microconidia are produced and efficiently dispersed under
wet conditions which predispose cassava plants to infection.
This probably explains why the disease could be very severe
under wet conditions.
Ayesu-Offei and Antwi-Boasiako—Production of Microconidia by Cercospora henningsii Allesch 657
Our results have suggested that production of microconidia occurred most abundantly when free water was
available. Since observations indicate that wet conditions
promote severe development of brown leaf spot of cassava,
integrated management strategies for the control of the
disease should exploit the drought-resistant properties of
cassava and encourage large-scale cultivation of the crop in
areas of low and moderate rainfall. Some spores of the
fungus may be dislodged from lesions in strong wind which
causes plants to sway, vibrate or rub against one another.
Inter-cropping will increase distances between cassava plants
and therefore reduce the incidence of dispersal of the spores
from plant to plant through water splash and contact
between plants.
LITERATURE CITED
Ayesu-Offei EN, Carter MV. 1971. Epidemiology of leaf scald of
barley. Australian Journal of Agricultural Research 22 : 383–390.
Ayesu-Offei EN, Clare BG. 1970. Processes in the infection of barley
leaves by Rhynchosporium secalis. Australian Journal of Biological
Sciences 23 : 299–307.
Ayres PG, Owen H. 1970. Factors influencing spore germination in
Rhynchosporium secalis. Transactions of the British Mycological
Society 54 : 389–394.
Bellotti AC, Van Schoonhoven A. 1978. Mite and insect pests of cassava.
Annual ReŠiew of Entomology 23 : 39–67.
Jameson JD. 1970. Agriculture in Uganda. 2nd edn. Oxford : Oxford
University Press, 116–276.
Leather RI. 1959. Diseases of economic plants in Ghana other than
cacao. Ghana Ministry of Food and Agriculture Bulletin 1.
Lozano JC, Booth RH. 1974. Diseases of cassava (Manihot esculenta
Crantz). Pans 20 : 30–54.
Pritchard NJ, Bell AA. 1967. Relative activity of germination inhibitors
from spores of smut and rust fungi. Phytopathology 57 : 932–938.
Solomon ME. 1952. Control of humidity with potassium hydroxide,
sulphuric acid or other solutions. Bulletin of Entomological
Research 42 : 543–554.
Talbot PHB. 1971. Principles of fungal taxonomy. London : Macmillan
Press.
Terry ER, Oyekan JO. 1976. Cassava diseases of Africa reviewed.
SPAN 19 : 116–118.