Phytopathologia Mediterranea
Firenze University Press
www.fupress.com/pm
The international journal of the
Mediterranean Phytopathological Union
Research Papers
Citation: A. Yirgu, A. Gezahgne, T.
Alemu, M. Havenga, L. Mostert (2021) First
report of Didymosphaeria rubi-ulmifolii
associated with canker and dieback
of apple trees in southern Ethiopia.
Phytopathologia Mediterranea 60(2):
229-236. doi: 10.36253/phyto-12400
Accepted: March 17, 2021
Published: September 13, 2021
Copyright: © 2021 A. Yirgu, A. Gezahgne, T. Alemu, M. Havenga, L. Mostert.
This is an open access, peer-reviewed
article published by Firenze University Press (http://www.fupress.com/pm)
and distributed under the terms of the
Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Data Availability Statement: All relevant data are within the paper and its
Supporting Information files.
Competing Interests: The Author(s)
declare(s) no conflict of interest.
Editor: Vladimiro Guarnaccia, DiSAFA
- University of Torino, Italy.
First report of Didymosphaeria rubi-ulmifolii
associated with canker and dieback of apple
trees in southern Ethiopia
Abraham YIRGU1,*, Alemu GEZAHGNE1, Tesfaye ALEMU2, Minette
HAVENGA3, Lizel MOSTERT3
1
Central Ethiopia Environment and Forest Research Center, P.O.Box 33042, Addis Ababa, Ethiopia
2 College of Natural and Computational Sciences, Addis Ababa University P.O.Box 1176,
Ethiopia
3 Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland,
7602, South Africa
*Corresponding author. E-mail address: abrahamyirguw@gmail.com
Summary. Cultivation of apple trees in the highlands of Ethiopia began in 1955. In
2014, blistering of the bark due to cankers on the main stems mostly below the grafting points, followed by dieback and eventually death of apple trees, was observed in
apple orchards in the Hadiya Zone in Ethiopia. This study aimed to identify the causal
agent of canker and dieback symptoms on the apple trees. Symptomatic trunks from
20 trees (ten per cultivar) were sampled. Isolations were performed from ten trunks
(five per cultivar). Fungus colonies with similar cultural features were obtained from
all the samples, and the morphology of a representative isolate was characterized. Phylogenetic analyses of the concatenated internal transcribed spacers 1 and 2 and 5.8S
rRNA gene, large subunit and actin gene regions confirmed the identity of two isolates as Didymosphaeria rubi-ulmifolii. Pathogenicity was confirmed for one isolate by
inoculations of healthy branches of ‘Anna’ and ‘Dorsett Golden’ apple trees resulting
in lesion formation, and subsequent re-isolation of the inoculated fungus. This study
is the first report of D. rubi-ulmifolii associated with dieback of apple trees. This pathogen caused death of more than 26% of apple trees in one commercial orchard, and
could cause severe losses for smallholder apple growers in Ethiopia. Future studies are
required to assess the magnitude, distribution and management options of this economically important canker disease in Ethiopia.
Keywords. Malus pumila, Paraconiothyrium brasiliense, stem canker.
INTRODUCTION
Malus pumila Mill. (Rosaceae; syn. M. domestica Borkh.) is native to
southwest Asia (Hedberg et al., 1989), and apple is the fourth most important
horticultural fruit crop in the world and is by far atypical temperate fruit
tree that can reach 8–12 m high (Hedberg et al., 1989; Tromp, 2005; BekelePhytopathologia Mediterranea 60(2): 229-236, 2021
ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.36253/phyto-12400
230
Abraham Yirgu et alii
Tesemma, 2007; FAO, 2010). Although originating and
common in temperate regions, apple trees are grown at
2,300 m above sea level in many tropical and subtropical regions, and have become an increasingly important
source of income in Ethiopia (Erez, 2000; Bekele Tesemma, 2007). Commercial apple cultivars are grafted on
seed grown rootstocks.
Around 1955, missionaries established apple
orchards (medium and high-chilling cultivars) in the
Chencha highlands of the Southern Nations and Nationalities People (SNNP) Regional state of Ethiopia (Fetena
et al., 2014). This region has minimum temperatures
of 11 to 13°C and maxima of 18 to 23°C, 900 to 1200
mm annual rainfall, and altitudes of 2300 to 3250 m
(Hailemichael, 2006). In recent decades, government and
non-government organizations have engaged in propagation of low-chilling grafted apple trees and rootstocks
from abroad to improve the livelihood and income of the
rural communities in this region. Approximately 138,
000 plants (105,000 grafted apples and 33,000 rootstocks)
were imported between 1998 to 2007 (Sisay, 2007).
As apple production has expanded, the impacts of
biotic agents have increased. Assessments in Chencha
and Bonke districts showed that apple scab, powdery
mildew, green aphids, scale insects and green plant bugs
were the most serious apple diseases and pests (Fetena
and Lemma, 2014; Fetena et al., 2014). In addition to
significant losses in the production from ‘Bond’s Red
Royal Gala’, ‘Crispin’ and ‘Jona Gold’, ‘ Royal Gala’ was
replaced by ‘Crispin’ in Chencha district, due to susceptibility of ‘Royal Gala’ to apple scab (Fetana and Lemma, 2014). White root rot, caused by Rosellinia necatrix
Berl. ex Prill., also caused death of mother apple trees
and grafted plants. The MM106 rootstock, which was
once considered as a superior rootstock for Chencha district, was found to be susceptible to crown rot on poorly
drained soils (Fetena et al., 2014).
In early 2014, extensive damage due to cankers and
dieback was observed on apple trees at Gibagri Farm,
located in SNNP Regional State in Ethiopia. The study
described here was carried out to identify the causal
agent of this disease.
MATERIALS AND METHODS
Sample collection, isolation and characterization of pathogenic fungus
The Foke orchard of Gibagri Farm PLC, is situated
at Olewa Peasant Association in Gibe District, Hadiya
Zone in SNNPR in Ethiopia, about 260 km from the
capital city Addis Ababa. The district has a wet Woina
Dega climate, with annual rainfall of 1100 mm, with
Nitosol reddish loamy and deeply structured soil (pH 5
to 6), and altitude of 2,000 m. Gibagri Farm PLC grows
the apple ‘Anna’, ‘Princesa’ and ‘Dorsett Golden’ imported from Spain in 2009 and 2012, on 14 ha of land. Field
survey was conducted in Foke Orchard in December
2015. Symptomatic trunk samples from 20 apple trees,
ten each from ‘Anna’ and ‘Dorsett Golden’ grafted on
rootstocks M7, MM111 and MM106, were systematically sampled and collected, as described by Cloete et al.
(2011). These samples were separately packed in paper
bags, labeled and brought to the Forest Protection Laboratory of the Central Ethiopia Environment and Forest
Research Center (CEE-FRC), Addis Ababa. In the laboratory, samples were kept at 4°C in a refrigerator before
further analysis.
Three to four segments of wood fragments were collected from the margins between necrotic and healthy
tissues of five symptomatic apple trunks per cultivar.
The fragments were excised into approx. 2×2 cm pieces.
These were then surface sterilized in 70% ethanol followed by 5% sodium hypochlorite, each for 2 min., and
then rinsed in sterilized distilled water. The tissue pieces
were then air-dried, each aseptically halved using a sterile knife, and then placed in 90 mm diam. Petri dishes
containing malt extract agar (MEA, Oxoid Ltd) amended with 100 mg of streptomycin sulphate. In addition,
cross-sections cut from stems showing disease symptoms on both sides were placed in 19.5 mm diam. Petri
dishes with moistened double-layered filter paper to
induce pycnidium formation. The filter papers were regularly monitored and moistened with distilled water. All
dishes were placed on a laboratory bench and incubated
in 12 h light and 12 h dark, at 20 to 25°C room temperature. Emerging mycelia and pycnidia were transferred to
potato dextrose agar (PDA; Oxoid Ltd.) amended with
streptomycin sulphate.
The growth characteristics of the isolated fungus
were determined based on mycelium growth rate and
colony colour on PDA after 7 d incubation at 25°C in
darkness. Pycnidium formation in a moist chamber was
also examined under a dissecting microscope. Sporulation of the isolated fungus was induced by transferring
mycelia from the margin of 7-d-old pure cultures onto
20 g L-1 water agar supplemented with sterilized pine
needles (Su et al., 2012). Based on morphological similarities, a fungus isolate obtained from ‘Anna’ was designated as AY-1 and an isolate from ‘Dorsett Golden’ was
designated as AY-2. Dimensions (length and width) and
shape of 50 conidia from the AY-1 isolate were measured
and described at 400× magnification using an Olympus BX 63 camera-mounted microscope. Conidiomata
231
First report of Didymosphaeria rubi-ulmifolii affecting apple trees in southern Ethiopia
were sectioned (10 µm thick) with a freeze microtome
and measured at 400× and 1000× magnification using a
Nikon Eclipse E600 compound microscope with a Nikon
DMX1200C digital camera attachment. Ten conidiomata
and 30 conidiogenous cells were measured. Cultures of
both isolates were deposited at the Ethiopian Biodiversity Institute and Forest Protection laboratory of CEEFRC.
Molecular characterization and phylogenetic analysis
DNA was extracted from 4-d-old cultures of isolates AY-1) and AY-2), and the regions were amplified
of the internal transcribed spacers 1 and 2 and 5.8S
rRNA (ITS), 28S rRNA the large subunit (LSU) and
the partial actin (ACT) genes. The primers used for
these regions were V9G/LS266 for ITS, LROR/LR6 for
LSU and ACT512F/ACT783R for ACT. The PCR conditions and sequencing were as described by Samson et
al. (2010), and were carried out at the Westerdijk Fungal Biodiversity Institute (CBS), AD Utrecht, the Netherlands. Consensus sequences were made from the forward and reverse sequences, and were lodged in GenBank (MK167444 to MK167448). For phylogenetic analysis, sequences generated by Ariyawansa et al. (2014) were
added as reference sequences. Kalmusia longisporum
CBS 582.83 was included as the out-group. Newly generated sequences of the three gene regions were aligned
separately using the E-INS-i algorithm in the MAFFT
plugin of Geneious R9 software (Katoh and Standley,
2013), visually inspected for obvious alignment errors,
and concatenated in Geneious. Maximum likelihood
analysis was performed in PhyML-mpi (Guindon et al.,
2010) under the best-fit model (HKY+I+G). Branch support was calculated from 100 bootstrap replicates for the
concatenated dataset.
Pathogenicity test
The AY-1 isolate was used in a pathogenicity test
conducted on apple trees at Foke apple orchard, in
November 2017. Four apple trees each of ‘Anna’ and
‘Dorsett Golden’ from different rows of the farm were
randomly selected for the inoculation trial. On each tree,
two healthy and oppositely situated branches with mean
diameter of 11 mm (range 10 to 14 mm) were selected.
One branch was inoculated with a mycelium plug of the
fungus and the other branch was treated with a sterile
PDA plug (as the negative control). In total eight branches were inoculated with the fungus and eight were treated with sterile PDA plugs. The methods of Luque et al.
(2006) and Sami et al. (2014) were adopted with modifications. The bark of each of the selected branches was
cleaned with 70% ethanol before inoculation. Holes
(4 mm diam.) were made in the branches using a cork
borer. Mycelium plugs (4 mm diam.) cut from colony
margins in 10-d-old PDA cultures were placed on the
wounds with mycelium facing the host cambium. A 4
mm diam. sterile PDA plug was similarly placed on one
of the alternative branches of each tree as the control
treatment. Inoculated wounds were wrapped with Parafilm® (American National Can) to prevent contamination
and desiccation of the inoculated areas. After 8 weeks,
all inoculated branches were destructively sampled and
brought to CEE-FRC Forest Protection laboratory. The
lengths of developed lesion’s development in the cambium of inoculated branches were measured after removing the bark of each branch surrounding the inoculation
point. Re-isolations from symptomatic cambium tissues
beyond the areas of inoculation were performed onto
PDA. ANOVA analysis of the lesion length data was
done using XLStat. Differences in mean lesion lengths
formed on the two cultivars were assessed with Fishers
least significant differences (LSD) at P ≤ 0.05.
RESULTS
Disease symptoms and severity in the field
Based on the documents available in Gibagri Farm
PLC, 31,500 grafted apple trees were imported from
Spain between May 2009 and July 2012. Of these, 384
trees were rejected due to their poor rooting and dry
wood. As well, 8,186 apple trees were eradicated from
the nursery and orchard sites at the farm up to November 2017, due to development of canker symptoms. The
trees showed blistering of the bark of the main stems
mostly below the grafting points, and eventually produced exudates, lanceolate leaf development, abscission
of blossom and fruits, and dieback that progressively led
to the death of the trees (Figure 1a). Cross-sectional cuts
of stems of symptomatic trees showed either circular or
triangular discolorations in the wood tissues (Figure 1b
and 1c).
Morphological characteristics of fungi isolated fungi on
PDA
Morphology of the fungi isolated from symptomatic
samples of the two host cultivars was the same. Colonies
on PDA were whitish with short woolly texture (Figure
2a). Black conidiomata formed on the incubated wood
232
Abraham Yirgu et alii
Figure 1. Apple tree at Foke Farm showing dieback symptoms (a) and main stem cross section symptoms (b and c).
samples, and fungus structures are illustrated in Figure
2. Conidiomata on PDA were superficial or immersed in
the agar, were dark brown to black, and were (-316)414659(-673) µm wide and (-303)494-584(-776) µm tall.
They were mostly single (sometimes multiple) and
clumped together (Figure 2b), with short necked ostioles.
And were 75–180 µm long and 87-256 µm wide at the
base. The conidiomata walls had textured outer layers
21–42 µm thick with thin, dark brown cells, and were
lined with inner layers of hyaline globose cells 16–32 µm
thick (Figure 2c to e). The surfaces of conidiomata walls
were covered with dark brown hyphae. Conidiogenous
cells were discrete or assembled into protruding masses, and were indeterminate and phialidic, formed from
the inner cells all over the conidiomata walls, and were
hyaline to pale yellow, and broadly ampulliform to globose (Figure 2f), with distinct periclinal thickening (Figure 2g). Phialide collarettes mostly absent, occasionally
with visible annelations, and measured (-3)4-6(-8) µm
× (-1.5)2-2.5(-3) µm (Figure 2h). Conidia were ellipsoidal to short-cylindrical, rounded at both ends, 1-celled,
olivaceous, and 3-4(-4.8) µm × (-1)1.5-2(-2.8) µm (Figure 2i). The morphological characteristics were similar to those described for Paraconiothyrium brasiliense
Verkley (Verkley et al., 2004; Paul and Lee, 2014). This
species was synonymised with Didymosphaeria rubiulmifolii Ariyawansa, Erio Camporesi & K.D. Hydeby
Ariyawansaet al. (2014b), based on phylogenetic analyses. The sexual stage as described by Ariyawansaet al.
(2014a) was not observed in the present study.
Molecular characterization and phylogenetic analysis of
fungus isolate
Phylogenetic analyses of the concatenated ITS, LSU
and ACT gene regions confirmed the identity of isolates
AY-1 and AY-2 as Didymosphaeria rubi-ulmifolii (Figure
3). Both isolates grouped together with D. rubi-ulmifolii
sensu stricto, with a 99% bootstrap support (CBS 100299,
the type strain of P. brasiliense). The D. rubi-ulmifolii
sensu lato subclade did not have significant support, only
an internal cluster of three D. rubi-ulmifolii sensu lato
isolates were associated with the present study’s two isolates (bootstrap support of 68%).
Pathogenicity test
Prominent brown lesions caused by the fungus after
inoculation were observed on the branches of ‘Anna’
and ‘Dorsett Golden’ apple trees (Figure 4). For ‘Anna’,
the lesions lengths were from 23 to 86 mm (mean = 54
mm; n = 4). For ‘Dorsett Golden’, the lesion lengths were
from 26 to 46 mm (mean =32 mm; n = 4). Mean lesion
First report of Didymosphaeria rubi-ulmifolii affecting apple trees in southern Ethiopia
233
Figure 2. Cultural and morphological structures of Didymosphaeria rubi-ulmifolii (isolate AY-1). After 7 d growth on PDA (a), conidiomata
with oozing conidia (b), cross section through a conidioma illustrating the neck (c), cross sections through conidioma wall (d and e), conidiogenous cells (f to h), arrow indicating periclinal thickening and conidia (i). Scale bars: in b = 1000 µm; c = 100 µm; d = 50 µm; e to i = 10 µm.
lengths for the two cultivars were not significantly different (P = 0.175). Two of the control branches (one
branch each from the two cultivars) had slightly pale
brown discolorations at the inoculation sites, which did
not exceed 4 mm in length, and no D. rubi-ulmifolii was
isolated from these lesions. Re-isolation of the same fungus (colony characteristics described above) from each of
the pathogen inoculated branches confirmed the pathogenicity of D. rubi-ulmifolii on apple trees.
DISCUSSION
This study has confirmed that the trunks of M.
pumila trees at Foke farm were infected by D. rubi-ulmifolii (syn. Paraconiothyrium brasiliense). This pathogen
caused gradual decline and dieback of apple trees and
decreased growth and production of fruit in the affected
orchards. The dieback caused by this pathogen was so
severe that the orchard owner decided to remove all of
the apple trees and plant a different crop.
Previous studies showed that D. rubi-ulmifolii has
been isolated from a number of plant species, including:
fruits of Coffea arabica in Brazil; Ginkgo biloba, Pinus
tabulaeformis and leaves of Pinus glauca in Canada;
Alliaria petiolata in the USA; marine fish in China; wetland surface water in Japan; discoloured wood of a living Platanus acerifolia tree in Italy; South African peach,
nectarine, and plum trees; and Chinese Maple leaves in
Korea (reviewed in Paul and Lee, 2014). Didymosphaeria
rubi-ulmifolii was reported to cause disease on one-yearold commercial apple trees and was also found causing latent infections in certified nursery trees in South
Africa (Havenga et al., 2019). The present paper is, therefore, the second report of canker disease on apple trees
caused by D.rubi-ulmifolii, and the first report in Ethiopia. The potential pathogenicity of D. rubi-ulmifolii on
detached apple shoots was shown by Cloete et al. (2011),
who inoculated the fungus isolated from pears onto
apple shoots, and showed it to be pathogenic causing significant lesions.
234
Abraham Yirgu et alii
Figure 3. Maximum likelihood phylogeny of the selected genera within Didymosphaeriaceae, as estimated from concatenated alignments
of the internal transcribed spacers 1 and 2 and 5.8S rRNA gene (ITS), the 28S rRNA large subunit gene (LSU) and the partial actin (ACT)
gene regions. Maximum likelihood bootstrap percentages are indicated at the nodes. Support values less than 50% bootstrap were omitted.
Kalmusia longisporum CBS 582.83 was included as the outgroup.
The main reasons behind the serious damage from
D. rubi-ulmifolii on apple trees may be a combination of
cultivar susceptibility and suitable environmental conditions (high air humidity and low soil pH) for the pathogen. Low soil pH causes stress to apple trees. Due to the
slow development of symptoms, the cause of the disease
was not initially attributed to fungal canker. Didymosphaeria rubi-ulmifolii was isolated from one farm and
the incidence of the fungus across other apple-growing
districts of Ethiopia has not been established.
In conclusion, in addition to the updating of quarantine measures required for the importation of trees
harbouring latent quarantine pathogen infections, future
studies are required to determine the magnitude, distribution and consequences of this pathogen on the cultivation and production of apple trees in Ethiopia. This
information will assist the livelihoods of the rural communities in different apple production of this country.
ACKNOWLEDGEMENTS
The authors thank Gibagri Farm PLC, for assisting
and covering expenses during fieldwork for this study. The
CEE-FRC allowed use of laboratory consumables, and the
Ethiopian Institute of Biodiversity (EIB) sent specimens to
The Netherlands. The Westerdijk Fungal Biodiversity Insti-
tute, AD Utrecht, the Netherlands provided fungus molecular characterization and identification. Prof. Peter Braun
and colleagues at the University of Geisenheim, Germany
assisted in this study. Ms. Mag. pharm. Hermine LotzWinter and her team at the Faculty of Biological Sciences
Department of Mycology, Biologicum, Goethe-University,
Germany and Dr. Ulrike Damm, Head of Section Mycology, Senckenberg Museum of Natural History, Görliz, Germany gave assistance with preparation of this paper. Tiruwork Tesfa and Almaz Asefa assisted during laboratory
work at CEE-FRC. Dr. Adane Ayele facilitated microscopy
at Armauer Hansen Research Institute (AHRI), Addis
Ababa, Ethiopia. The assistance of the Editor of the Journal and two anonymous reviewers to improve the manuscript is also gratefully acknowledged.
LITERATURE CITED
Ariyawansa H.A., Camporesi E., Thambugala K.M.,
Mapook A., Kang J., … Hyde K.D., 2014a. Confusion surrounding Didymosphaeria—phylogenetic and
morphological evidence suggest Didymosphaeriaceae
is not a distinct family. Phytotaxa 176: 102–119. doi:
10.11646/phytotaxa.176.1.12.
Ariyawansa H.A., Tanaka K., Thambugala K.M.,
Phookamsak R., Tian Q., Hyde K.D., 2014b. A
First report of Didymosphaeria rubi-ulmifolii affecting apple trees in southern Ethiopia
Figure 4. Brown lesion caused by Didymosphaeria rubi-ulmifolii
(isolate AY-1) on a branch of apple ‘Anna’, and non-inoculated (control) branch, 8 weeks post inoculation.
molecular phylogenetic reappraisal of the Didymosphaeriaceae (=Montagnulaceae). Fungal Diversity 68:
69–104. doi: 10.1007/s13225–014-0305-6.
Bekele-Tesemma A., 2007. Useful trees and Shrubs for
Ethiopia: Identification, Propagation and Management
for 17 Agroclimatic zones. World Agroforestry Centre,
East Africa Region, Nairobi, Kenya, 552 pp.
Cloete M., Fourie P.H., Damm U., Crous P.W., Mostert
L., 2011. Fungi associated with dieback symptoms of
apple and pear trees, a possible inoculum source of
grapevine trunk disease pathogens. Phytopathologia
Mediterranea 50: S176–S190. doi: 10.14601/Phytopathol_Mediterr-9004.
Erez A., 2000. Bud dormancy; phenomenon, problems
and solutions in the tropics and subtropics. In Tem-
235
perate Fruit Crops in Warm Climates (A. Erez, ed.),
Kluwer Academic Publishers, The Netherlands,
17–48.
FAO, 2010. FAOSTAT: statistics database. Food and Agriculture Organization of the United Nations (FAO).
Available at: http://www.apps.fao.org/.
Fetena S., Lemma B., 2014. Assessment on major apple
diseases and insect pests in Chencha and Bonke
Woredas of Gamo Gofa Zone, Southern Ethiopia.
Scholarly Journal of Agricultural Science 4: 394–402.
Fetena S., Shara S., Anjulo A., Gulie G., Woldesenbet F.,
Yilma B., 2014. Survey on apple production and variety identification in Chencha District of Gamo Gofa
Zone, Southern Ethiopia. Journal of Agricultural Food
Technology 4: 7–15.
Guindon S., Dufayard J.F., Lefort V., Anisimova M.,
Hordijk W., Gascuel O., 2010. New algorithms and
methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology 59; 307–321. doi: 10.1093/sysbio/
syq010.
Havenga M., Gatsi G.G., Halleen F., Spies C.F.J., van der
Merwe R., Mostert L., 2019. Canker and wood rot
pathogens present in young apple trees and propagation material in the Western Cape of South Africa.
Plant Disease 103: 3129–3141. doi: 10.1094/PDIS04–19-0867-RE.
Hailemichael B.G., 2006. Impact of male out-migration
on rural women’s livelihood: the case of Chencha
woreda, South Ethiopia. MSc Thesis, Addis Ababa
University, Addis Ababa, Ethiopia, 120 pp.
Hedberg O.,1989. Pittosporaceae to Araliaceae. In: Flora
of Ethiopia and Eritrea. (I. Hedberg, S. Edwards, ed.),
The National Herbarium, Addis Ababa, Ethiopia, and
Department of Systematic Botany, Uppsala, Sweden,
732.
Katoh K., Standley D.M., 2013. MAFFT multiple
sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30, 772–780. doi: 10.1093/molbev/
mst010.
Luque J., Sierra D., Torres E., Garcia F., 2006. Cryptovalsa ampelina on grapevines in N.E. Spain: identification and pathogenicity. Phytopathologia Mediterranea
45: S101–S109. doi: 10.14601/Phytopathol_Mediterr-1838.
Paul N.C., Lee H.B., 2014. First Record of Endophytic
Paraconiothyrium brasiliense isolated from Chinese
Maple Leaves in Korea. Korean Journal of Mycology
42: 349–352. doi: 10.4489/KJM.2014,42.4.349.
Sami S., Mohammadi H., Heydarnejad J., 2014. Phaeoacremonium species associated with necrotic wood
236
of pome fruit trees in Iran. Journal of Plant Pathology
96: 487–495. doi: 10.4454/JPP.V9613.006.
Samson R.A., Houbraken J., Thrane U., Frisvad J.C.,
Andersen B., 2010. CBS Laboratory manual series 2,
Food and Indoor Fungi. CBS-KNAW Fungal Biodiversity Centre Utrecht, The Netherlands, 390 pp.
Sisay A., 2007. Ethiopia-GTZ Promotes apples in the
highlands of Ethiopia. Available at: www.nazret.com/
news (Accessed November 10, 2017).
Su Y.Y., Qi Y.L., Cai L., 2012. Induction of sporulation in
plant pathogenic fungi. Mycology 3(3), 195–200.
Tromp J., 2005. Fruit ripening and quality. In: Fundamentals of Temperate Zone Tree Fruit Production (J.
Tromp, A.D. Webster, S.J. Wertheimeds, ed.), Backhuys Publishers, Leiden, The Netherlands, 295–310.
Verkley G.J.M., da Silva M., Wicklow D.T., Crous P.W.,
2004. Paraconiothyrium, a new genus to accommodate the mycoparasite Coniothyrium minitans,
anamorphs of Paraphaeosphaeria, and four new species. Studies in Mycology 50: 323–335.
Abraham Yirgu et alii