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"International Symposium
on Current Trends in Plant Protection"
2S - 28111 September, 2012
Belgrade, Serbia
PROCEEDINGS
MHCTMyYT 3a 3awTMTY 6Mlba M)KMBOTHY cpセmhy@
M3 6eorpa,.a
Institute for Plant Protection and Environment, Belgrade
Ro itza Rodeva. Ilija Karov, Zornitsa Stoyanova •...
International Symposium: Current Trends in Plant Protection
Proceedings
257
UDK: 633.842-24(497.7)
PHOMOPSIS CAPSICIAND COLLErOrHICHl/M COCCOIPES
INFECTING PEPPER IN MACEDONIA
1
2
2
ROSSITZA RODEVA , ILilA KAROV • ZORNITSA STOYANOVA I, BllJANA KOVACEVIK ,
V ASILISSA MANOV A', RALITSA GEORGIEV A'
phaseolina. O.
Gerais.
, J., Simpson. J.
(phaseolus vilgaris
drought stress.
lA. (2007):
of pathegenicity.
Diseases and
resistance in
76.646-649.
with chlorate
Ilnstitute of Plant Physiology and Genetics, 1113 Sofia. Bulgaria
2Goce Delcev University-Stip. Macedonia
Phomopsis capsici and Colletotrichum coccodes were found on pepper fruits during a joint
expedition carried out in Macedonia. The lesions caused by P. capsici often occurred together and
resembled slightly those incited by C. coccodes. Phomopsis lesions could be differentiated on the
basis of pliable leathery condition of the affected tissue and of pycnidium presence while C. coccodes
produced lesions with regular round shape and abundant acervuli. setae and microsclerotia in
coloni1-ed fruit tissue. On some fruits P. capsici caused single infection but mixed infections of
Phomopsis and Colletotrichum were observed. as well. C. coccodes is a soil-borne pathogen that
produces long-lasting structures (microsclerotia) in the plant debris. The development of this
pathogen on pepper might contribute to the building up of inoculum in the soil which could serve as
reservoir for other Solanaceae. To our knowledge, this is the first report of P. capsici and C. coccodes
on pepper in Macedonia.
Variations of
hem Iran. Australian
Key words: Capsicum annuum, Colletotrichum coccodes, pepper anthracnose, Phomopsis
capsici, fruit decay
prijatnimi uslovijami
INTRODUCTION
12 (I). University of
Last years, Phomopsis capsici (Magnaghi) Sacc and several Colletotrichum 'pp.
(Colletotrichum gloeosporioides (Penz.) Penz. & Saccardo in Penz., C. acutatum
Simmonds ex Simmonds and C. coccodes (WaUr.) SJ. Hughes) occurred on pepper in
Bulgaria with increasing frequency (Rodeva et aI., 2009a; 2009b; 2009c), In August 2011 a
joint expedition was carried out in Macedonia related to the implementation of ERA 226
project. Two new pepper fungal pathogens were found, isolated, described and
characterized. The results are presented in this paper.
,peratures and mineral
rophomina phaseolina
Biological Control of
X'ugoslavia. September
s of mineral nutrition.
(in Serbian).
lnicijevic. M. (2011):
eetroot in 2010. Plant
;ugar beet root rot in
2, Brussels. Belgium,
MATERIAL AND METHODS
Initial isolations from diseased pepper fruits on potato dextrose agar (PDA) revealed
the presence of P. capsici and C. coccodes. Four Macedonian (MK26.1, MK26.2, MK7.1,
MK7.2) and one Bulgarian (B8.1) isolates of C. coccodes were selected for the investigations. Identification of Colletotrichum spp. was performed on the basis of morphological
and cultural characteristics (conidial size and morphology, colony morphology and growth
258
Phomopsis capsici and Colletotrichum coccoides infecting pepper in Macedonia
rate, presence or absence of: teleomorph, setae, microsclerotia) (Sutton, 1992; Freeman et
aI., 1998; Tozze Jr. et aI., 2006) and pathogenicity tests. Growth rate and colony appearance
were studied on three nutrient media: PDA, 0.2% malt extract agar (MEA) and oatmeal
agar (OA), which were inoculated with mycelial discs taken from the edge of growing
colonies. For the pathogenicity tests the isolates were grown on PDA. Pin pricked detached
pepper fruits were inoculated with agar plugs containing fungal mycelium. Control fruits
were inoculated with sterile PDA discs. Tomato and eggplant fruits were additionally
inoculated with C. coccodes for comparison. Fruits were incubated for 7 days at 25°C under
100% relative humidity. Reisolations were made at the end of the experiments. At leasl 100
conidia of each isolate were measured on the images with Carnoy program.
Total DNA of investigated Colletotrichum isolates was extracted directly from
mycelium by DNeasy Plant mini kit (Qiagen, Hilden, Germany). PCR amplifications were
performed with both Colletotrichum-specific primer set CciFlICc2RI and C. coccodesspecific nested primers CclNFlICc2NRl. The gels were visualized by UV
transillumination, their electronic images were taken by ImageQuant ISO imager (GE
Healthcare) and densitometrically analyzed with ImageQuantTL7 software (GE Healthcare)
to determine the approximate length of the resulting PCR products.
RossilZa Rodeva, I
The C. coccodes colonie
rate was recorded on OA
lowest - on MEA (39.0±7.6 mm)
Macedonian ones on all nutrient
ro e nuance mainly in the
great number of microsclerotia
RESULTS
C. coccodes was isolated mainly from fruits, seeds of heavily infected fruits and
occasionally from roots although it could infect stems and leaves. The disease symptoms
were observed in the area of Kochani (village Dolni Pod log) on variety Kurtov ka kapija
and in Strurnica (village Bosilovo) also in the postharvest period. Fruit anthracnose
appeared first as small, circular, slightly sunken lesions on the surface of ripening fruits
(Fig. la). Majority of infections were observed on ripe or over-ripe fruits. The spots quickly
enlarged in concentric circles, coalesced, become deeply sunken with dark brown border
and developed a water-soaked appearance directly beneath the skin (epidermis) of the fruit
(Fig. I b). At first small rounded acervuli containing rose conidial mass were developed on
the surface and beneath the lesion (Fig. Ic,e). Later then the fungus formed small, dark
survival structures called sclerotia (Fig. Id).
Fig. 2. CoLLetotrichum coccodes:
malt extract agar (b) and oatmeal
Fig. 1. Colletotrichum coccodes: Symptoms of anthracnose on pepper fruits, early infection (a);
coalescent lesions (b); young lesions with sporulating acervuli (c); fully developed Ie ions with
rnicrosclerotia (d); sporulating acervuli (e)
Rossitza Rodeva, I1ija Karov, Zomitsa Stoyanova, ...
259
The C. coccodes colonies were slowly growing. On the ninth day the high t growth
rate was recorded on OA (49.5±5.l mm) and PDA (47.8±4.8 mm) (Fig. 2c,a) and the
lowest - on MEA (39.0±7.6 mm) (Fig. 2b). Bulgarian isolate had higher growth rate than
Macedonian ones on all nutrient media used in the study. The colony colour w gray with
ro e nuance mainly in the periphery, where acervuli with conidia developed. With aging a
great number of microsclerotia appeared under mycelium.
Fig. 2. Colletotrichum coccodes: Appearance of 10 days old colonies potato dextrose agar (a).
malt ex.tract agar (b) and oatmeal agar (c) (left and middle column Macedonian isolates, right
column - Bulgarian isolate)
260
Phomopsis capsici and Colletotrichum coccoides infecting pepper in Macedonia
Conidia were hyaline, straight, cylindrical, aseptate with two to seven oil globules
measuring (19.2) 21.3±1.7 (24.6) x (3.1) 4.l±OA (4.7) 11m (Fig. 3a). Acervuli with elae
longer than 100 11m developed (Fig. 3b).
Ros iua Rodeva, IIija Karov,
PCR amplification with
of -450 bp in all isolates
(Cullen at aI., 2002) (Fig. SA).
single specific PCR band of
containing as a template DNA
Fig. 3. Colletotrichllm coccodes: Conidia (a) and acervulus with conidiophores, conidia and
setae (b) (Scale bars = 10 11m)
All investigated C. coccodes isolates were pathogenic for pepper, tomato and
eggplant (Fig. 4a,b,c). Water-soaked circular lesions appeared three days after in eulalion
(dai) that became soft and slightly sunken. Wet, gelatinous conidi.aI mass from fungal
fruiting bodies (acervuli) gradually covered the lesions. About IO - 14 dai the central lesion
part darkened where abundant microsc1erotia developed.
Fig. 4. Colletotrichwn coccodes: Symptoms on artificially inoculated pepper (a), tomato (b) and
eggplant (c) fruits - 14 days after inoculation
Fig. 5. Molecular identification of
with primers CclFlICc2RI ; gel
CcINFI/Cc2NRI; Lanes 2-13 (C.
MK7.l, MK7.2 and C. sp. isolates
water); lanes I:
Ro itza Rodeva, l\ija Karov, Zornitsa Stoyanova, ...
261
peR amplification with genus-specific primers (CcIFlICc2RI) gave a single band
of -4S0 bp in all isolates analyzed (c. coccodes and C. sp.) as expected from the literature
(Cullen at aI., 2002) (Fig. SA). However, with the nested primer set CcINFlICc2NRI, a
single specific PCR band of expected size (-3S0bp) was obtained only in those reactions
containing as a template DNA from investigated C. coccodes isolates (Fig. S8).
and
Fig. S. Molecular identification of different Colletotrichllm species: Gel (A): PCR amplification
with primers CclFIICc2RI; gel (B): PCR amplification with C. coeeodes-specific primers
CcINFl/Cc2NR I; Lanes 2-13 (c. coccodes isolates B8.1, B2.1, B40.la, MK26.1, MK26.2,
MK7.1. MK7.2 and C. sp. isolates B27, BI.I, B29,S2, S3); lanes 14: Negative controls (mQ
water); lanes 1: DNA marker GeneRuler I kb+ (Fermentas).
262
Phomopsis capsid and Colletotrichwn coccoides infecting pepper in Macedonia
P. capsici was found in the village Zubovo, Strumica region, on pepper fruits cv.
Zubovska kapija (domestic pepper variety of Kurtovska kapija, which is grown only in this
village). Until now P. capsici was not recorded on pepper anywhere else in the country.
The symptoms of P. capsici on the fruits appeared as brown rot extending in wavy rings
more rapidly longitudinally than laterally in the tissue (Fig. 6a). Infection progress led to
fruit decay. The dead tissue became dry and bleached in the centre where black globose to
subglobose subepidermal or erumpent pycnidia were noticed. White felt-like mycelium
developed inside the damaged fruits. The fungus was i olated not only from pericarp but
also from seeds of disea ed pepper fruits.
some fruits P. capsid
CoLletotrichum were observed
To our knowledge, this
Macedonia. Recently. C.
a\., 2008) . C. coecodes is an
structures (microsclerotia) in
includes pepper, tomato and
The outbreak of this pathogen
the soil serving as an important
Financial support of
acknowledged .
Fig. 6. Phomopsis capsid: symptoms (a), colony morphology (b) and alpha and beta conidia (c)
P. capsici de eloped fast growing colonies on PDA (Fig. 6b). They were initially
white, later developrng light to dark brown patches and reached the Petri dish border' 7
day after inoculatio n. S mall bla k pycnidia (150-250 セュI@
devel ped after 10-12 days. The
extruded conidia were vi ible as y lIo wi h globo. e sli me. The reverse s ides of the colonies
were grayi h with darker regions coinciding with o nidiomata. The i olates produced
abundan t al pha conidia - urUcellular. traight. ovoid to oblo ng-fusoid, hyaline, biguttulate,
with averrage dimension 6.8 x 2.9 1ffi1. scarce beta conidia which are unicellular, curved or
hamate, e guttulate with averrage dimensions 29. 8 x 1. J..lm and very rarely gamma conidia
- unicellular, traight, paddle hape. multiguttulate. with averrage dimensions 11.0 x 2.9
Ilm (Fig. 6c). The first o ne nly are viable and infective. No perithecia were found on the
over win ter d di eased pepper fruits or in ullure. Artificial inoculation of detached pepper
fru its led to ucce fu l infection ten day after inoculation.
DISCUSSION
Anthracnose of pepper caused by C. coccodes appeared to be a devastating di ease
of ripe fruits cau ing severe damages to both field and post harvest levels in warm and
rainy seasons. The infections occurred on green fruits but symptoms were visible after the
ripening . During the season the pathogen was spread from infected to healthy fruits with
conidia splashed by rain. overhead irrigation, or by picking fruit from wet plants. The
lasting structures called sclerotia could survive in soil for up to three years and 」 。オ セ@
infections either directly or by producing secondary spores. The lesions caused by P.
capsici often occurred together and resembled slightly those resulting from infection by C.
coecodes. Phomopsis lesions could be differentiated on the basis of pliable leathery
condition of the affected tissue and of pycnidium presence while C. coeeodes produced
lesions with regular round shape and abundant microsclerotia in colonized fruit tissue. On
CuUen. D. W .• Lees, A. K..
coccodes from soil and
Plant Pathology, 51: 281
Freeman. S.• Katan, T. Shabi. E.
for anthracnose diseases
Rodeva. R.. Pandeva, R.,
caused by Phomopsis
Rodeva. R., Stoyanova, Z.,
caused by Colletotrichum
562.
Rodeva, R .• Stoyanova, Z.,
pepper fruits in Bulgaria.
Sulton. B. C. (1992) : The
Colletotrichum. Biology,
CAB lnt!.. Wallingford,
Tozze Jr., H. J .• Mello, M. B. A..
characterization of
Phytopathologica, 32: 71 TrkuJja. V. , StojCic. 1., Brkljat. G.,
mogucnosti njegova sUlbijar
I Macedon ia
Rossitza Rodeva, Ilija Karov, Zornitsa Stoyanova, ...
'er fruits cv.
only in this
the country.
wavy ri ngs
Igress led to
( globose to
セ@ mycelium
;>ericarp but
some fruits P. capsici caused single infection but mixed infections of Phomopsis and
Colletotrichum were observed, as well.
To our knowledge, this is the first report of C. coccodes and P. capsici on pepper in
Macedonia. Recently, C. coccodes has been reported in Bosnia and Herzegovina (Trkulja et
aI., 2(08). C. coccodes is an important soil-borne pathogen that produces long-lasting
structures (microsclerotia) in the dying plant parts, with host range in Solanaceae that
includes pepper, tomato and eggplant, causing anthracnose and potato, causing black-dot.
The outbreak of this pathogen on pepper can lead to an enrichment of inoculum density in
the soil serving as an important source of inoculum for other solanaceous crops.
セイ・@
ACKNOWLEDGEMENTS
Financial support of SEE-ERA.NET PLUS project ERA 226 is gratefully
acknowledged.
REFERENCES
conidia (c)
initially
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he colonies
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