First report of Didymosphaeria rubi-ulmifolii associated with canker and dieback of apple trees in southern Ethiopia

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.


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;Bekele-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 . 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;. 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 .
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.

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 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 CEE-FRC.

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 Gen-Bank (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 Para-film® (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.

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).

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 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. 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.