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Causal agents of powdery mildew on Moringa stenopetala (Baker f.) cuf. and Moringa oleifera lam. in Ethiopia
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Causal agents of powdery mildew on Moringa stenopetala (Baker f.) cuf. and Moringa oleifera lam. in Ethiopia a a,*, Ludmila Holkova a, Ivana Safr nkova a, Petr Ne mecb Marie Bartíkova 1665/1, 613 00 Brno, Czechia Department of Crop Science, Breeding and Plant Medicine, Faculty of AgriSciences, Mendel University in Brno, Zemedelska 1665/1, 613 Department of Forest Botany, Dendrology and Geobiocoenology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemedelska 00 Brno, Czechia
a
b
A R T I C L E
I N F O
Article History: Received 30 March 2019 Revised 3 October 2019 Accepted 9 December 2019 Available online xxx Edited by AR NdhlalaI Keywords: Leveillula taurica Erysiphe aquilegiae Cabbage-tree Drumstick-tree
A B S T R A C T
Moringa Adans. is a tropical plant genus belonging to the plant family Moringaceae, consisting of 13 species, all naturally growing and some of these cultivated in the tropics and subtropics. Species of Moringa are multipurpose trees of significant economic importance with vital nutritional, industrial, and medicinal applications. Therefore, the health of the trees is of utmost importance, but there is no methodology for its protection. The objective of the work was to investigate the spectrum of pathogens of the order Erysiphales (powdery mildews) on leaves of Moringa stenopetala (Baker f.) Cuf. and Moringa oleifera Lam. in the district (woreda) Arba Minch Zuria in Ethiopia, growing on a plantation and on native, freely growing trees in the area. Morphological examination and molecular PCR assay were used to determine powdery mildews from v.) G. Arnaud on Moringa stenopetala Moringa spp. Results show the presence of pathogen Leveillula taurica (Le on the plantation as well as on the leaves of freely growing trees. On Moringa oleifera, PCR analysis supported the presence of the pathogen Erysiphe aquilegiae DC. Additionally, the symptoms of the pathogens were described for possible control management in intensive production of Moringa plants. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction The monogenic plant family Moringaceae, with Moringa as a sole genus, includes 13 deciduous tree species (Jeffrey and Mabberley, 2007), from which the most known are Moringa oleifera, Moringa stenopetala and Moringa peregrina (Forssk.) Fiori. M. stenopetala is a highly valued native tree to northern Kenya and southern Ethiopia (Abuye et al., 2003), where moringa leaves are used in people’s meals, daily. M. oleifera is a native tree to sub-Himalayan tracts of northern India (Jahn, 1991) and was introduced to Ethiopia over the last decade (Melesse et al., 2012). M. oleifera is a highly esteemed tree used for multiple purposes: a potential biofuel (Mofijur et al., 2014), a natural coagulant for water treatment (Camacho et al., 2017) and mainly used as an important food commodity, valued for its nutrition content and its medical purposes (Anwar et al., 2007). In May 2017 a plantation of M. stenopetala and M. oleifera was established for the first time in Ethiopia, in the district of Arba Minch Zuria, State Southern Nations, Nationalities and People’s regional state (SNNPR), where M. stenopetala and M. oleifera grow in monoculture.
* Corresponding author. E-mail addresses: [email protected] (M. Bartíkova), ), [email protected] [email protected] (L. Holkova ankova ), [email protected] (P. Ne mec). (I. Safr
Moringa spp. are considered to be a species resistant to most pests and common diseases (Mridha and Barakah, 2017). Nevertheless, severe defoliation may be caused by the caterpillar of the moth Noorda blitealis Walker on M. stenopetala in Ethiopia (Bedane et al., 2013). Fungal pathogens such as Pseudocercospora morindicola, Cercospora spp., Sphaceloma morindae, Puccinia moringae and Polyporus gilvus were reported to cause leaf-spots on M. oleifera (Janick and Paull, 2008). Further fungi (Aspergillus niger, A. flavus, Alternaria alternata, Fusarium oxysporum, Macrophomina phaseolina and Rhizopus stolonifera) were identified on pods of the same species (Mridha and Barakah, 2017). In addition to these fungi, powdery mildews Leveillula taurica (Ullasa and Rawal, 1984) and Pseudoidium moringae (Hosag.) U. Braun & R.T.A. Cook (bas. Oidium moringae Hosag.) were described on Moringa oleifera (Braun and Cook, 2012). Powdery mildews are common obligatory biotrophic ascomycetes of the order Erysiphales (Sinclair and Lyon, 2005). They are easily recognizable by a white powdery cover on the plant surface, but it is difficult to identify and classify them, especially only according the anamorph stage of reproduction (Cunnington et al., 2003). The aim of this work was to identify and determine the powdery mildews on M. stenopetala and M. oleifera growing on the plantation in Arba Minch Zuria Woreda, Ethiopia. The data may serve for future control management, especially with regards to possible introduction of the pathogen from M. oleifera to M. stenopetala.
https://doi.org/10.1016/j.sajb.2019.12.002 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
et al., Causal agents of powdery mildew on Moringa stenopetala (Baker f.) cuf. and Moringa oleifera lam. Please cite this article as: M. Bartíkova in Ethiopia, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.002
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2. Material and methods 2.1. Sample collection and preparation Leaves of Moringa stenopetala and Moringa oleifera infected with powdery mildew were sampled at the plantation located 18,5 km north of Arba Minch in Arba Minch Zuria Woreda, Southern Ethiopia (6.1147722 N, 37.6118119E) and from trees of M. stenopetala freely growing in the region. The plantation extends in an area of 30 £ 70,5 m, trees spaced 1,5 m within a row, with 1 m distance between rows. In total, 705 trees of each species were planted. The region was visited in 2018 during the vegetation period (Mar 19th and Jun 5th) and each time 30 samples of compounded leaves were collected from 15 randomly selected infected trees of M. stenopetala and M. oleifera on the plantation and 30 samples from 8 freely growing trees of M. stenopetala in the area from 2 km north to 45 km south from Arba Minch. Fresh leaves were stored for one day in plastic bags and transported in a cooling box to the lab, where they were placed in a refrigerator (at 4 °C) for 3 days before analysis. Part of freshly collected leaves was immediately placed between pressed paper sheets to be dried and transported as preserved herbarium specimens to the laboratory. 2.2. Morphological determination Powdery mildews on conserved infected leaves were determined by morphological characteristics in a laboratory environment. Transparent tape (tesa SE, Germany) was used on dried leaves to obtain the mycelium with conidiophores and conidia. An Olympus BX41 microscope (Olympus Corporation, Japan) with a 200 500 £ magnification was used for microscopic determination of the pathogens. Measuring of morphological characteristics was conducted in 65 repetitions using an Olympus C-5050 Zoom Digital Camera (Olympus Corporation, Japan) and QuickPHOTO CAMERA software (PROMICRA, s.r.o., CZ). Obtained measurements were then compared with published data (Gorter and Eiker, 1987; Braun and Cook, 2012). Based on the morphological determination, samples of powdery mildew on M. stenopetala from a natural habitat and from the plantation were found to be identical, therefore only samples from the plantation were subjected to molecular assay. 2.3. DNA isolation
identification of the pathogen by sequencing analysis. A BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., USA) was used for fluorescence-based cycle sequencing of ITS fragments, mixed and sequenced according to the standard protocol. After performing the cycle sequencing, the mixtures were purified by using a BigDye XTerminator Purification Kit (Thermo Fisher Scientific Inc., USA). Samples were analysed using a 3500 Series Genetic Analyzer (Thermo Fisher Scientific Inc., USA). To identify the pathogens, acquired sequences were submitted and compared with registered ITS sequences available at GenBank, using NCBI BLAST (online available database, National Center for Biotechnology Information, U.S. National Library of Medicine, USA). A fragment of sequence of MO was aligned with other sequences registered in GenBank identical from 99,4 100% by MAFFT version 7 software, using the average linkage (UPGMA) method to obtain a phylogenetic tree. Sequences marked as “uncultured sequences of Erysiphe” in GenBank were not included in our analysis.
3. Results It was possible to observe symptoms of the disease caused by powdery mildews with the naked eye on leaves of Moringa stenopetala and Moringa oleifera on the plantation, as well on the leaves of native M. stenopetala trees. The typical appearance of a powdery mildew, white layers of mycelia and conidiophores with conidia, were visible on the surface of the leaves. Mycelium of powdery mildew on the lower side of the leaves of M. stenopetala was dense (Fig. 1), initially restricted by leaf nervature and then coalescing together. In later stages of the disease, the pathogen caused yellow discoloration, deformation, and necrosis of the leaves and premature defoliation (Fig. 2). Mycelium of powdery mildew on M. oleifera creating radial patches on the upper side of the leaves, was less dense in comparison to the pathogen on M. stenopetala and patches of mycelium merged together. No chasmothecia of powdery mildews were observed on M. stenopetala and M. oleifera on the plantation, nor in the natural habitat. 3.1. Morphological determination Mycelium of powdery mildew on Moringa stenopetala was internal, with conidiophores emerging from stomata, individually or in
Actively growing powdery mildews from the fresh leaves of M. stenopetala (MS) and M. oleifera (MO) were used for DNA isolation. Fungal material, mainly conidiophores with conidia, was scraped directly from the surface of the leaves and the DNA of the pathogen was isolated according to the standard protocol using DNeasy Plant Mini Kit (Qiagen, Germany). The concentration of the isolated DNAs was quantified by spectrophotometry (Life Science Analyzer: BioMate 3 Spectrophotometer, Thermo Fisher Scientific Inc., USA). 2.4. DNA sequencing PCR amplification of a single fragment of a partial sequence of the 18S DNA gene, complete sequence of ITS1, 5.8S DNA, ITS2, and a partial sequence of the 28S DNA gene, was conducted by forward primer PMITS1 (50 -TCGGACTGCCCTAGGGAGA-30 ) and reverse primer PMITS2 (50 -TCACTCGCCGTTACTGAGGT-30 ) according to Cunnington et al. (2003). The thermal cycling profile consisted of an initial denaturation step. 35 cycles of 1minute denaturation at 94 °C, 1 min annealing at 60 °C, 2 min extension at 72 °C and the final extension step for 10 min at 72 °C using a TGradient Thermocycler (Biometra, Analytik Jena, Germany). Obtained amplified DNAs (730 bp) were purified by QIAquickÒ PCR Purification Kit (Qiagen, Germany) according to the standard protocol and subsequently used for the
Fig. 1. White patches of powdery mildew on abaxial side of leaves of M. stenopetala.
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Moringa oleifera (sample MO) as Erysiphe aquilegiae DC. or Pseudoidium moringae (Hosag.) U. Braun & R.T.A. Cook. 3.2. Molecular determination Acquired PCR products of powdery mildew pathogens, conducted in ITS region with primers PMITS1/PMITS2, were 770 bp long, proving the presence of pathogens of the order Erysiphales. By molecular sequence analysis of these products, MS was identified as Leveillula taurica (97,2%; sequence registered in GenBank as MN108158). The sequence of MO (registered in GenBank as MN108157) corresponds from 100% with Erysiphe aquilegiae (KY653202.1, KY653167.1, KY653166.1) and with registered sequence of Erysiphe buhrii (KY653205.1). Followed by a 99,9% match to E. circaeae (KY660882.1). Phylogenetic analysis (Fig. 7) revealed our sequence to be more closely related to the group of sequences of Erysiphe aquilegiae (KY653202.1, KY653166.1, KY653167.1). Fig. 2. White patches of powdery mildew on adaxial side of M. stenopetala leaves.
4. Discussion groups (Fig. 3). Conidiophores were simple, occasionally branched, straight. Primary and secondary conidia were produced on the terminal of conidiophores, in different shapes, ellipsoid, cylindrical or clavate (Figs. 4, and 5). By microscopic methods and morphological v (Table 1). characteristics, MS was identified as Leveillula taurica Le Hyphae of pathogen sample MO were non-septate, hyphal appressoria were lobed and solitary (Fig. 6). Conidiophores arose from the upper surface of mother cells, erect, forming single, ellipsoid conidia on the terminal. Foot cells were often slightly curved-sinuous at the bases, followed by one cell. Observed morphological characteristics are presented in Table 2. Based on the morphological characteristics, it was not possible to strictly classify the pathogen present on
Tested Moringa species grow on the plantation in Arba Minch Zuria in immediate proximity to each other. Nevertheless, different species of powdery mildew were detected on each of the plants. Leveillula taurica (anamorph Oidiopsis sicula Scal.), present on Moringa stenopetala, is widely distributed polyphage in subtropical and tropical areas of Africa, Asia, southern parts of Europe, and southern North to South America. It can infect plants of more than 74 families (Palti, 1988). Braun & Cook (2012) present, that is has been described on various species of family such as Brassicaceae, Euphorbiaceae, Papaveraceae, Ranunculaceae, Rosaceae and Solanaceae. It has been published by Parotta (2009), that pathogen L. taurica is known to cause in India severe infection on papaya (Carica papaya L.), also a member of
Fig. 3. Group of conidiophores of MS, identified as Leveillula taurica.
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Fig. 4. Dimorphic conidia of MS, identified as Leveillula taurica.
Fig. 5. Germinating conidium of MS, identified as Leveillula taurica.
Table 1 Morphological characteristics of powdery mildew on Moringa stenopetala (Baker f.) Cuf.
hyphae (diameter, mm) hyphal appressoria (length, mm) foot-cells conidiophores (length, mm) conidia (length £ diameter mm)
Leveillula taurica (Braun and Cook, 2012)
Leveillula taurica (Gorter and Eicker, 1987)
MS
2 8 Nippled shaped, lobed to multilobed, coralloid
(2,5 )4( 6) multilobed straight >200 (50 )60 70( 80) £ (13,7 )17,5 20( 23,7)
3 6
120 300 £ 4 7 50 80 £ (9-)12 16( 20) secondary: (50 )55 75( 80) £ 11 16( 20)
straight 109 229 51 76 £ 16 21
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Fig. 6. Appressorium of sample MO. Table 2 Morphological characteristics of powdery mildews on Moringa oleifera Lam.
hyphae (diameter, mm) hyphal appressoria (length, mm) foot-cells foot-cells (length £ diameter, mm) following cells conidiophores (length, mm) conidia (length £ diameter, mm)
Erysiphe aquilegiae (Braun and Cook, 2012)
Pseudoidium moringae (Braun and Cook, 2012, p. 602)
MO
5 7 3 7, lobed straight/slightly curved-sinuous (15 )20 40 £ 7 11 (0 )1 2( 3)
3,5 7,5 lobed straight 20 30 £ 7,5 9,5 1 70 90 26,5 45,5 £ 11,5 19
6 12 (4 ) 7 ( 10), lobed straight/slightly curved-sinuous 26 34 £ 7 9 1 48 88 27 38 £ 12 20
(25 )28 50 £ (12) 16 22( 24)
Fig. 7. A phylogenetic tree gained by maximum likelihood analysis of assessed fragment ITS sequence supported by Bootstrap analysis. MO sequence is under the code MN108157.
et al., Causal agents of powdery mildew on Moringa stenopetala (Baker f.) cuf. and Moringa oleifera lam. Please cite this article as: M. Bartíkova in Ethiopia, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.002
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order Brassicales. As Ethiopia is one of the top ten papaya producers in the world (Fuentes and Santamaría, 2014), it is important to know, that the presence of L. taurica on M. stenopetala can have a negative impact on the health stage of papaya produced in the area. To date, this pathogen has not been described on Moringa stenopetala. However, Moringa oleifera is known to be susceptible to L. taurica (Ullasa and Rawal, 1984), though no traces of L. taurica were detected on leaves of M. oleifera. This survey was conducted only one year after the plantation was established, therefore more data need to be collected and assessed in following years, to determine whether the pathogen L. taurica will spread also to M. oleifera. Based only on the morphological characteristics (lengths of the conidiophore, conidia formation, size and shape of the foot cells and conidia, and germination of conidia) of the anamorph state of the powdery mildew isolated from Moringa oleifera, it was not possible to classify the isolate of the pathogen into a species. Comparing the morphological characteristics of our isolate to the morphological characteristics presented by Braun & Cook (2012), pathogens Pseudoidium moringae and Erysiphe aquilegiae were considered as possible matches (Table 2). No teleomorph stage of MO was observed, which makes its cs and Jankovics, 2011). According to identification more difficult (Kova the molecular analysis followed by phylogenetic analysis, the classification of the pathogen as Erysiphe aquilegiae was accepted, despite the fact that the phylogenetic analysis shows allocation of E. aquilegiae into two groups, as is presented also by Cunnington et al. (2004). Detailed analysis of read sequences by MAFFT discovered, that division of Erysiphe aquilegiae sequences into two groups is probably a consequence of one single mutation (InDel.) in position 45 (according to KY 653204.1). The finding corresponds with results published by Kovacs & Jankovics (2011), who identified one to five nucleotide differences in the ITS sequences within one species of Erysiphales. Nevertheless, it was not possible to include the sequence of pathogen Pseudoidium moringae for phylogenetic comparison, as this pathogen has not been registered in GenBank, but the most related Pseudoidium spp. (MG654732.1, LC342963.1, LC342966.1) and Oidium sp. differ from our sequence in 7 mutations on 1000 bp. M. oleifera is described by Braun & Cook (2012) as the only host of P. moringae in India, while the host range of E. aquilegiae covers various genera of family Ranunculaceae, Gentianaceae and Magnoliaceae, distributed in North America, all of Europe, the Causasus and having been introduced to Australia, New Zealand, South Africa and South America. However, the occurrence of powdery mildew on plants outside of their usual host range is possible (Liberato and Cunningrton, 2006). Next to the powdery mildew, the pathogen Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei was occasionally present on the leaves of Moringa stenopetala. This pathogen is one of the fungi causing leafspots and has been described on Moringa oleifera in Guam (Dixon et al., 2009). Presence of pests and pathogens on Moringa stenopetala trees growing in nature may be a possible source for monocultures of Moringa spp. on plantations. It is possible that weather conditions and the microclimate of the plantation might lead to increased distribution of pests and pathogens in coming years. Considering the use of Moringa spp. trees, monitoring of its health and effective control measures need to be addressed, especially in expanding intensive production. Declaration of Competing Interest
Acknowledgment This research was financially supported by a project of Czech Republic Development Cooperation no. CRA 02/2016/03 "Implementation of holistic management and climate smart agriculture in the Baso river catchment, Arba Minch Zuria Woreda, SNNPR, Ethiopia". Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2019.12.002. References Abuye, C., Urga, K., Knapp, H., Selmar, D., Omwega, A.M., Imungi, J.K., Winterhalter, P., 2003. A compositional study of Moringa stenopetala leaves. East African Medical Journal 80 (5), 51–56. Anwar, F., Latif, S., Ashraf, M., Gilani, A.H., 2007. Moringa oleifera: a food plant with multiple medicinal uses. Phytotherapy Research 21, 17–25. https://doi.org/ 10.1002/ptr.2023. Braun, U., Cook, R.T.A., 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS-KNAW Fungal Biodiversity Centre. Bedane, T.M., Singh, S.K., Selvaraj, T., Negeri, M., 2013. Distribution and damage of moringa moth (Noorda blitealis walker) on Moringa stenopetala baker (Cufod.) in Southern rift valley of Ethiopia. Journal of Agricultural Technology 9 (4), 963–985. Camacho, F.P., Sousa, V.S., Bergamasco, R., Ribau Teixeira, M., 2017. The use of Moringa oleifera as a natural coagulant in surface water treatment. Chemical Engineering Journal 313, 226–237. https://doi.org/10.1016/j.cej.2016.12.031. Cunnington, J.H., Lawrie, A.C., Pascoe, I.G., 2004. Unexpected ribosomal DNA internal transcribed spacer sequence variation within Erysiphe aquilegiae sensu lato. Fungal Diversity 16, 1–10. Cunnington, J.H., Takamatsu, S., Lawrie, A.C., Pascoe, I.G., 2003. Molecular identification of anamorphic powdery mildews (Erysiphales). Australasian Plant Pathology 32 (3), 421–428. https://doi.org/10.1071/AP03045. Dixon, L.J., Schlub, R.L., Penezny, K., Datnoff, L.E., 2009. Host specialization and phylogenetic diversity of corynespora cassiicola. Phytopathology 99, 1015–1027. Fuentes, G., Santamaría, J.M., 2014. Papaya (Carica papaya L.): Origin, domestication, and production. In: Ming, R., Moore, P. (Eds.), Genetics and Genomics of Papaya. Plant Genetics and Genomics: Crops and Models. Springer, New York, pp. 3–15. Gorter, G.J.M.A., Eicker, A., 1987. Additional first records of perfect stages of some powdery mildew fungi in South Africa, including a new species. South African Journal of Botany 53 (1), 93–97. https://doi.org/10.1016/s0254-6299(16)31479-x. Janick, J., Paull, R.E., 2008. The encyclopedia of fruit and nuts. CABI(ISBN 978-0-85199638-7). Jahn, S.A.A., 1991. The traditional domestication of a multipurpose tree Moringa stenopetala (Bak. f.) cuf. in the Ethiopian rift valley. Ambio 20 (6), 244–247. Jeffrey, C., Mabberley, D.J., 2007. The plant book. KEW Bulletin 43 (4), 722–724. https:// doi.org/10.2307/4129975. cs, G.M., Jankovics, T., 2011. Variation in the nrDNA its sequences of some powKova dery mildew species: do routine molecular identification procedures hide valuable information? European Journal of Plant Pathology 131, 135–141. Liberato, J.R., Cunnington, J.H., 2006. First record of Erysiphe aquilegiae on a host outside the Ranunculaceae. Australasian Plant Pathology 35, 291–292. Melesse, A., Steingass, H., Boguhn, J., Schollenberger, M., Rodehutscord, M., 2012. Effects of elevation and season on nutrient composition of leaves and green pods of Moringa stenopetala and Moringa oleifera. Agroforestry Systems 86 (3), 505– 518. Mofijur, M., Masjuki, H.H., Kalam, M.A., Atabani, A.E., Arbab, M.I., Cheng, S.F., Gouk, S.W., 2014. Properties and use of Moringa oleifera biodiesel and diesel fuel blends in a multi-cylinder diesel engine. Energy Conversion and Management 82, 169–176. https://doi.org/10.1016/j.enconman.2014.02.073. Mridha, M.A.U., Barakah, F.N., 2017. Diseases and pests of moringa: a mini review. Acta Horticulturae 1158,. https://doi.org/10.17660/ActaHortic.2017.1158.14 117-12. 4. Palti, J., 1988. The Leveillula mildews. The Botanical Review 54 (4), 423–535. https:// doi.org/10.1007/BF02858418. Parrotta, J.A., 2009. Moringa oleifera LAM., 1785. In: Roloff, A., Weisgerber, H., Lang, U., €die Der Holzgew€achse: Handbuch Und Atlas Der DenStimm, B (Eds.), Enzyklopa drologie. WILEY-VCH Verlag GmbH & Co, KGaA, Weinheim, pp. 1–8. Sinclair, W.A., Lyon, H.H., 2005. Diseases of Trees and Shrubs, 2nd edn Cornell University Press, Ithaca, NY, USA, p. 660. Ullasa, B.A., Rawal, R.D., 1984. Papaver rhoeas and Moringa oleifera, two new hosts of papaya powdery mildew. Current Science 53, 754–755.
None.
et al., Causal agents of powdery mildew on Moringa stenopetala (Baker f.) cuf. and Moringa oleifera lam. Please cite this article as: M. Bartíkova in Ethiopia, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.002
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South African Journal of Botany 000 (2019) 1 6
Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Causal agents of powdery mildew on Moringa stenopetala (Baker f.) cuf. and Moringa oleifera lam. in Ethiopia a a,*, Ludmila Holkova a, Ivana Safr nkova a, Petr Ne mecb Marie Bartíkova 1665/1, 613 00 Brno, Czechia Department of Crop Science, Breeding and Plant Medicine, Faculty of AgriSciences, Mendel University in Brno, Zemedelska 1665/1, 613 Department of Forest Botany, Dendrology and Geobiocoenology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemedelska 00 Brno, Czechia
a
b
A R T I C L E
I N F O
Article History: Received 30 March 2019 Revised 3 October 2019 Accepted 9 December 2019 Available online xxx Edited by AR NdhlalaI Keywords: Leveillula taurica Erysiphe aquilegiae Cabbage-tree Drumstick-tree
A B S T R A C T
Moringa Adans. is a tropical plant genus belonging to the plant family Moringaceae, consisting of 13 species, all naturally growing and some of these cultivated in the tropics and subtropics. Species of Moringa are multipurpose trees of significant economic importance with vital nutritional, industrial, and medicinal applications. Therefore, the health of the trees is of utmost importance, but there is no methodology for its protection. The objective of the work was to investigate the spectrum of pathogens of the order Erysiphales (powdery mildews) on leaves of Moringa stenopetala (Baker f.) Cuf. and Moringa oleifera Lam. in the district (woreda) Arba Minch Zuria in Ethiopia, growing on a plantation and on native, freely growing trees in the area. Morphological examination and molecular PCR assay were used to determine powdery mildews from v.) G. Arnaud on Moringa stenopetala Moringa spp. Results show the presence of pathogen Leveillula taurica (Le on the plantation as well as on the leaves of freely growing trees. On Moringa oleifera, PCR analysis supported the presence of the pathogen Erysiphe aquilegiae DC. Additionally, the symptoms of the pathogens were described for possible control management in intensive production of Moringa plants. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction The monogenic plant family Moringaceae, with Moringa as a sole genus, includes 13 deciduous tree species (Jeffrey and Mabberley, 2007), from which the most known are Moringa oleifera, Moringa stenopetala and Moringa peregrina (Forssk.) Fiori. M. stenopetala is a highly valued native tree to northern Kenya and southern Ethiopia (Abuye et al., 2003), where moringa leaves are used in people’s meals, daily. M. oleifera is a native tree to sub-Himalayan tracts of northern India (Jahn, 1991) and was introduced to Ethiopia over the last decade (Melesse et al., 2012). M. oleifera is a highly esteemed tree used for multiple purposes: a potential biofuel (Mofijur et al., 2014), a natural coagulant for water treatment (Camacho et al., 2017) and mainly used as an important food commodity, valued for its nutrition content and its medical purposes (Anwar et al., 2007). In May 2017 a plantation of M. stenopetala and M. oleifera was established for the first time in Ethiopia, in the district of Arba Minch Zuria, State Southern Nations, Nationalities and People’s regional state (SNNPR), where M. stenopetala and M. oleifera grow in monoculture.
* Corresponding author. E-mail addresses: [email protected] (M. Bartíkova), ), [email protected] [email protected] (L. Holkova ankova ), [email protected] (P. Ne mec). (I. Safr
Moringa spp. are considered to be a species resistant to most pests and common diseases (Mridha and Barakah, 2017). Nevertheless, severe defoliation may be caused by the caterpillar of the moth Noorda blitealis Walker on M. stenopetala in Ethiopia (Bedane et al., 2013). Fungal pathogens such as Pseudocercospora morindicola, Cercospora spp., Sphaceloma morindae, Puccinia moringae and Polyporus gilvus were reported to cause leaf-spots on M. oleifera (Janick and Paull, 2008). Further fungi (Aspergillus niger, A. flavus, Alternaria alternata, Fusarium oxysporum, Macrophomina phaseolina and Rhizopus stolonifera) were identified on pods of the same species (Mridha and Barakah, 2017). In addition to these fungi, powdery mildews Leveillula taurica (Ullasa and Rawal, 1984) and Pseudoidium moringae (Hosag.) U. Braun & R.T.A. Cook (bas. Oidium moringae Hosag.) were described on Moringa oleifera (Braun and Cook, 2012). Powdery mildews are common obligatory biotrophic ascomycetes of the order Erysiphales (Sinclair and Lyon, 2005). They are easily recognizable by a white powdery cover on the plant surface, but it is difficult to identify and classify them, especially only according the anamorph stage of reproduction (Cunnington et al., 2003). The aim of this work was to identify and determine the powdery mildews on M. stenopetala and M. oleifera growing on the plantation in Arba Minch Zuria Woreda, Ethiopia. The data may serve for future control management, especially with regards to possible introduction of the pathogen from M. oleifera to M. stenopetala.
https://doi.org/10.1016/j.sajb.2019.12.002 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
et al., Causal agents of powdery mildew on Moringa stenopetala (Baker f.) cuf. and Moringa oleifera lam. Please cite this article as: M. Bartíkova in Ethiopia, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.002
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2. Material and methods 2.1. Sample collection and preparation Leaves of Moringa stenopetala and Moringa oleifera infected with powdery mildew were sampled at the plantation located 18,5 km north of Arba Minch in Arba Minch Zuria Woreda, Southern Ethiopia (6.1147722 N, 37.6118119E) and from trees of M. stenopetala freely growing in the region. The plantation extends in an area of 30 £ 70,5 m, trees spaced 1,5 m within a row, with 1 m distance between rows. In total, 705 trees of each species were planted. The region was visited in 2018 during the vegetation period (Mar 19th and Jun 5th) and each time 30 samples of compounded leaves were collected from 15 randomly selected infected trees of M. stenopetala and M. oleifera on the plantation and 30 samples from 8 freely growing trees of M. stenopetala in the area from 2 km north to 45 km south from Arba Minch. Fresh leaves were stored for one day in plastic bags and transported in a cooling box to the lab, where they were placed in a refrigerator (at 4 °C) for 3 days before analysis. Part of freshly collected leaves was immediately placed between pressed paper sheets to be dried and transported as preserved herbarium specimens to the laboratory. 2.2. Morphological determination Powdery mildews on conserved infected leaves were determined by morphological characteristics in a laboratory environment. Transparent tape (tesa SE, Germany) was used on dried leaves to obtain the mycelium with conidiophores and conidia. An Olympus BX41 microscope (Olympus Corporation, Japan) with a 200 500 £ magnification was used for microscopic determination of the pathogens. Measuring of morphological characteristics was conducted in 65 repetitions using an Olympus C-5050 Zoom Digital Camera (Olympus Corporation, Japan) and QuickPHOTO CAMERA software (PROMICRA, s.r.o., CZ). Obtained measurements were then compared with published data (Gorter and Eiker, 1987; Braun and Cook, 2012). Based on the morphological determination, samples of powdery mildew on M. stenopetala from a natural habitat and from the plantation were found to be identical, therefore only samples from the plantation were subjected to molecular assay. 2.3. DNA isolation
identification of the pathogen by sequencing analysis. A BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., USA) was used for fluorescence-based cycle sequencing of ITS fragments, mixed and sequenced according to the standard protocol. After performing the cycle sequencing, the mixtures were purified by using a BigDye XTerminator Purification Kit (Thermo Fisher Scientific Inc., USA). Samples were analysed using a 3500 Series Genetic Analyzer (Thermo Fisher Scientific Inc., USA). To identify the pathogens, acquired sequences were submitted and compared with registered ITS sequences available at GenBank, using NCBI BLAST (online available database, National Center for Biotechnology Information, U.S. National Library of Medicine, USA). A fragment of sequence of MO was aligned with other sequences registered in GenBank identical from 99,4 100% by MAFFT version 7 software, using the average linkage (UPGMA) method to obtain a phylogenetic tree. Sequences marked as “uncultured sequences of Erysiphe” in GenBank were not included in our analysis.
3. Results It was possible to observe symptoms of the disease caused by powdery mildews with the naked eye on leaves of Moringa stenopetala and Moringa oleifera on the plantation, as well on the leaves of native M. stenopetala trees. The typical appearance of a powdery mildew, white layers of mycelia and conidiophores with conidia, were visible on the surface of the leaves. Mycelium of powdery mildew on the lower side of the leaves of M. stenopetala was dense (Fig. 1), initially restricted by leaf nervature and then coalescing together. In later stages of the disease, the pathogen caused yellow discoloration, deformation, and necrosis of the leaves and premature defoliation (Fig. 2). Mycelium of powdery mildew on M. oleifera creating radial patches on the upper side of the leaves, was less dense in comparison to the pathogen on M. stenopetala and patches of mycelium merged together. No chasmothecia of powdery mildews were observed on M. stenopetala and M. oleifera on the plantation, nor in the natural habitat. 3.1. Morphological determination Mycelium of powdery mildew on Moringa stenopetala was internal, with conidiophores emerging from stomata, individually or in
Actively growing powdery mildews from the fresh leaves of M. stenopetala (MS) and M. oleifera (MO) were used for DNA isolation. Fungal material, mainly conidiophores with conidia, was scraped directly from the surface of the leaves and the DNA of the pathogen was isolated according to the standard protocol using DNeasy Plant Mini Kit (Qiagen, Germany). The concentration of the isolated DNAs was quantified by spectrophotometry (Life Science Analyzer: BioMate 3 Spectrophotometer, Thermo Fisher Scientific Inc., USA). 2.4. DNA sequencing PCR amplification of a single fragment of a partial sequence of the 18S DNA gene, complete sequence of ITS1, 5.8S DNA, ITS2, and a partial sequence of the 28S DNA gene, was conducted by forward primer PMITS1 (50 -TCGGACTGCCCTAGGGAGA-30 ) and reverse primer PMITS2 (50 -TCACTCGCCGTTACTGAGGT-30 ) according to Cunnington et al. (2003). The thermal cycling profile consisted of an initial denaturation step. 35 cycles of 1minute denaturation at 94 °C, 1 min annealing at 60 °C, 2 min extension at 72 °C and the final extension step for 10 min at 72 °C using a TGradient Thermocycler (Biometra, Analytik Jena, Germany). Obtained amplified DNAs (730 bp) were purified by QIAquickÒ PCR Purification Kit (Qiagen, Germany) according to the standard protocol and subsequently used for the
Fig. 1. White patches of powdery mildew on abaxial side of leaves of M. stenopetala.
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Moringa oleifera (sample MO) as Erysiphe aquilegiae DC. or Pseudoidium moringae (Hosag.) U. Braun & R.T.A. Cook. 3.2. Molecular determination Acquired PCR products of powdery mildew pathogens, conducted in ITS region with primers PMITS1/PMITS2, were 770 bp long, proving the presence of pathogens of the order Erysiphales. By molecular sequence analysis of these products, MS was identified as Leveillula taurica (97,2%; sequence registered in GenBank as MN108158). The sequence of MO (registered in GenBank as MN108157) corresponds from 100% with Erysiphe aquilegiae (KY653202.1, KY653167.1, KY653166.1) and with registered sequence of Erysiphe buhrii (KY653205.1). Followed by a 99,9% match to E. circaeae (KY660882.1). Phylogenetic analysis (Fig. 7) revealed our sequence to be more closely related to the group of sequences of Erysiphe aquilegiae (KY653202.1, KY653166.1, KY653167.1). Fig. 2. White patches of powdery mildew on adaxial side of M. stenopetala leaves.
4. Discussion groups (Fig. 3). Conidiophores were simple, occasionally branched, straight. Primary and secondary conidia were produced on the terminal of conidiophores, in different shapes, ellipsoid, cylindrical or clavate (Figs. 4, and 5). By microscopic methods and morphological v (Table 1). characteristics, MS was identified as Leveillula taurica Le Hyphae of pathogen sample MO were non-septate, hyphal appressoria were lobed and solitary (Fig. 6). Conidiophores arose from the upper surface of mother cells, erect, forming single, ellipsoid conidia on the terminal. Foot cells were often slightly curved-sinuous at the bases, followed by one cell. Observed morphological characteristics are presented in Table 2. Based on the morphological characteristics, it was not possible to strictly classify the pathogen present on
Tested Moringa species grow on the plantation in Arba Minch Zuria in immediate proximity to each other. Nevertheless, different species of powdery mildew were detected on each of the plants. Leveillula taurica (anamorph Oidiopsis sicula Scal.), present on Moringa stenopetala, is widely distributed polyphage in subtropical and tropical areas of Africa, Asia, southern parts of Europe, and southern North to South America. It can infect plants of more than 74 families (Palti, 1988). Braun & Cook (2012) present, that is has been described on various species of family such as Brassicaceae, Euphorbiaceae, Papaveraceae, Ranunculaceae, Rosaceae and Solanaceae. It has been published by Parotta (2009), that pathogen L. taurica is known to cause in India severe infection on papaya (Carica papaya L.), also a member of
Fig. 3. Group of conidiophores of MS, identified as Leveillula taurica.
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Fig. 4. Dimorphic conidia of MS, identified as Leveillula taurica.
Fig. 5. Germinating conidium of MS, identified as Leveillula taurica.
Table 1 Morphological characteristics of powdery mildew on Moringa stenopetala (Baker f.) Cuf.
hyphae (diameter, mm) hyphal appressoria (length, mm) foot-cells conidiophores (length, mm) conidia (length £ diameter mm)
Leveillula taurica (Braun and Cook, 2012)
Leveillula taurica (Gorter and Eicker, 1987)
MS
2 8 Nippled shaped, lobed to multilobed, coralloid
(2,5 )4( 6) multilobed straight >200 (50 )60 70( 80) £ (13,7 )17,5 20( 23,7)
3 6
120 300 £ 4 7 50 80 £ (9-)12 16( 20) secondary: (50 )55 75( 80) £ 11 16( 20)
straight 109 229 51 76 £ 16 21
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Fig. 6. Appressorium of sample MO. Table 2 Morphological characteristics of powdery mildews on Moringa oleifera Lam.
hyphae (diameter, mm) hyphal appressoria (length, mm) foot-cells foot-cells (length £ diameter, mm) following cells conidiophores (length, mm) conidia (length £ diameter, mm)
Erysiphe aquilegiae (Braun and Cook, 2012)
Pseudoidium moringae (Braun and Cook, 2012, p. 602)
MO
5 7 3 7, lobed straight/slightly curved-sinuous (15 )20 40 £ 7 11 (0 )1 2( 3)
3,5 7,5 lobed straight 20 30 £ 7,5 9,5 1 70 90 26,5 45,5 £ 11,5 19
6 12 (4 ) 7 ( 10), lobed straight/slightly curved-sinuous 26 34 £ 7 9 1 48 88 27 38 £ 12 20
(25 )28 50 £ (12) 16 22( 24)
Fig. 7. A phylogenetic tree gained by maximum likelihood analysis of assessed fragment ITS sequence supported by Bootstrap analysis. MO sequence is under the code MN108157.
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order Brassicales. As Ethiopia is one of the top ten papaya producers in the world (Fuentes and Santamaría, 2014), it is important to know, that the presence of L. taurica on M. stenopetala can have a negative impact on the health stage of papaya produced in the area. To date, this pathogen has not been described on Moringa stenopetala. However, Moringa oleifera is known to be susceptible to L. taurica (Ullasa and Rawal, 1984), though no traces of L. taurica were detected on leaves of M. oleifera. This survey was conducted only one year after the plantation was established, therefore more data need to be collected and assessed in following years, to determine whether the pathogen L. taurica will spread also to M. oleifera. Based only on the morphological characteristics (lengths of the conidiophore, conidia formation, size and shape of the foot cells and conidia, and germination of conidia) of the anamorph state of the powdery mildew isolated from Moringa oleifera, it was not possible to classify the isolate of the pathogen into a species. Comparing the morphological characteristics of our isolate to the morphological characteristics presented by Braun & Cook (2012), pathogens Pseudoidium moringae and Erysiphe aquilegiae were considered as possible matches (Table 2). No teleomorph stage of MO was observed, which makes its cs and Jankovics, 2011). According to identification more difficult (Kova the molecular analysis followed by phylogenetic analysis, the classification of the pathogen as Erysiphe aquilegiae was accepted, despite the fact that the phylogenetic analysis shows allocation of E. aquilegiae into two groups, as is presented also by Cunnington et al. (2004). Detailed analysis of read sequences by MAFFT discovered, that division of Erysiphe aquilegiae sequences into two groups is probably a consequence of one single mutation (InDel.) in position 45 (according to KY 653204.1). The finding corresponds with results published by Kovacs & Jankovics (2011), who identified one to five nucleotide differences in the ITS sequences within one species of Erysiphales. Nevertheless, it was not possible to include the sequence of pathogen Pseudoidium moringae for phylogenetic comparison, as this pathogen has not been registered in GenBank, but the most related Pseudoidium spp. (MG654732.1, LC342963.1, LC342966.1) and Oidium sp. differ from our sequence in 7 mutations on 1000 bp. M. oleifera is described by Braun & Cook (2012) as the only host of P. moringae in India, while the host range of E. aquilegiae covers various genera of family Ranunculaceae, Gentianaceae and Magnoliaceae, distributed in North America, all of Europe, the Causasus and having been introduced to Australia, New Zealand, South Africa and South America. However, the occurrence of powdery mildew on plants outside of their usual host range is possible (Liberato and Cunningrton, 2006). Next to the powdery mildew, the pathogen Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei was occasionally present on the leaves of Moringa stenopetala. This pathogen is one of the fungi causing leafspots and has been described on Moringa oleifera in Guam (Dixon et al., 2009). Presence of pests and pathogens on Moringa stenopetala trees growing in nature may be a possible source for monocultures of Moringa spp. on plantations. It is possible that weather conditions and the microclimate of the plantation might lead to increased distribution of pests and pathogens in coming years. Considering the use of Moringa spp. trees, monitoring of its health and effective control measures need to be addressed, especially in expanding intensive production. Declaration of Competing Interest
Acknowledgment This research was financially supported by a project of Czech Republic Development Cooperation no. CRA 02/2016/03 "Implementation of holistic management and climate smart agriculture in the Baso river catchment, Arba Minch Zuria Woreda, SNNPR, Ethiopia". Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2019.12.002. References Abuye, C., Urga, K., Knapp, H., Selmar, D., Omwega, A.M., Imungi, J.K., Winterhalter, P., 2003. A compositional study of Moringa stenopetala leaves. East African Medical Journal 80 (5), 51–56. Anwar, F., Latif, S., Ashraf, M., Gilani, A.H., 2007. Moringa oleifera: a food plant with multiple medicinal uses. Phytotherapy Research 21, 17–25. https://doi.org/ 10.1002/ptr.2023. Braun, U., Cook, R.T.A., 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS-KNAW Fungal Biodiversity Centre. Bedane, T.M., Singh, S.K., Selvaraj, T., Negeri, M., 2013. Distribution and damage of moringa moth (Noorda blitealis walker) on Moringa stenopetala baker (Cufod.) in Southern rift valley of Ethiopia. Journal of Agricultural Technology 9 (4), 963–985. Camacho, F.P., Sousa, V.S., Bergamasco, R., Ribau Teixeira, M., 2017. The use of Moringa oleifera as a natural coagulant in surface water treatment. Chemical Engineering Journal 313, 226–237. https://doi.org/10.1016/j.cej.2016.12.031. Cunnington, J.H., Lawrie, A.C., Pascoe, I.G., 2004. Unexpected ribosomal DNA internal transcribed spacer sequence variation within Erysiphe aquilegiae sensu lato. Fungal Diversity 16, 1–10. Cunnington, J.H., Takamatsu, S., Lawrie, A.C., Pascoe, I.G., 2003. Molecular identification of anamorphic powdery mildews (Erysiphales). Australasian Plant Pathology 32 (3), 421–428. https://doi.org/10.1071/AP03045. Dixon, L.J., Schlub, R.L., Penezny, K., Datnoff, L.E., 2009. Host specialization and phylogenetic diversity of corynespora cassiicola. Phytopathology 99, 1015–1027. Fuentes, G., Santamaría, J.M., 2014. Papaya (Carica papaya L.): Origin, domestication, and production. In: Ming, R., Moore, P. (Eds.), Genetics and Genomics of Papaya. Plant Genetics and Genomics: Crops and Models. Springer, New York, pp. 3–15. Gorter, G.J.M.A., Eicker, A., 1987. Additional first records of perfect stages of some powdery mildew fungi in South Africa, including a new species. South African Journal of Botany 53 (1), 93–97. https://doi.org/10.1016/s0254-6299(16)31479-x. Janick, J., Paull, R.E., 2008. The encyclopedia of fruit and nuts. CABI(ISBN 978-0-85199638-7). Jahn, S.A.A., 1991. The traditional domestication of a multipurpose tree Moringa stenopetala (Bak. f.) cuf. in the Ethiopian rift valley. Ambio 20 (6), 244–247. Jeffrey, C., Mabberley, D.J., 2007. The plant book. KEW Bulletin 43 (4), 722–724. https:// doi.org/10.2307/4129975. cs, G.M., Jankovics, T., 2011. Variation in the nrDNA its sequences of some powKova dery mildew species: do routine molecular identification procedures hide valuable information? European Journal of Plant Pathology 131, 135–141. Liberato, J.R., Cunnington, J.H., 2006. First record of Erysiphe aquilegiae on a host outside the Ranunculaceae. Australasian Plant Pathology 35, 291–292. Melesse, A., Steingass, H., Boguhn, J., Schollenberger, M., Rodehutscord, M., 2012. Effects of elevation and season on nutrient composition of leaves and green pods of Moringa stenopetala and Moringa oleifera. Agroforestry Systems 86 (3), 505– 518. Mofijur, M., Masjuki, H.H., Kalam, M.A., Atabani, A.E., Arbab, M.I., Cheng, S.F., Gouk, S.W., 2014. Properties and use of Moringa oleifera biodiesel and diesel fuel blends in a multi-cylinder diesel engine. Energy Conversion and Management 82, 169–176. https://doi.org/10.1016/j.enconman.2014.02.073. Mridha, M.A.U., Barakah, F.N., 2017. Diseases and pests of moringa: a mini review. Acta Horticulturae 1158,. https://doi.org/10.17660/ActaHortic.2017.1158.14 117-12. 4. Palti, J., 1988. The Leveillula mildews. The Botanical Review 54 (4), 423–535. https:// doi.org/10.1007/BF02858418. Parrotta, J.A., 2009. Moringa oleifera LAM., 1785. In: Roloff, A., Weisgerber, H., Lang, U., €die Der Holzgew€achse: Handbuch Und Atlas Der DenStimm, B (Eds.), Enzyklopa drologie. WILEY-VCH Verlag GmbH & Co, KGaA, Weinheim, pp. 1–8. Sinclair, W.A., Lyon, H.H., 2005. Diseases of Trees and Shrubs, 2nd edn Cornell University Press, Ithaca, NY, USA, p. 660. Ullasa, B.A., Rawal, R.D., 1984. Papaver rhoeas and Moringa oleifera, two new hosts of papaya powdery mildew. Current Science 53, 754–755.
None.
et al., Causal agents of powdery mildew on Moringa stenopetala (Baker f.) cuf. and Moringa oleifera lam. Please cite this article as: M. Bartíkova in Ethiopia, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.002