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1First Report of Neoerysiphe aloysiae Causing Powdery Mildew on Lemon Verbena
2(Aloysia citrodora) in Mexico
3A. R. Solano-Báez, Universidad Popular Autónoma del Estado de Puebla, Decanato de
4 Ciencias Biológicas, Facultad de Agronomía, 72410, Puebla, Puebla, Mexico; A. Venegas-
5Portilla, Universidad Autónoma Chapingo, Departamento de Parasitología Agrícola,
6 Texcoco, 56230, Estado de México, Mexico; J. Rodríguez-Mendoza, Dirección General de
7 Sanidad Vegetal, Centro Nacional de Referencia Fitosanitaria, Tecámac, 55740,
8 Estado de México, Mexico; M. Camacho-Tapia, Universidad Autónoma Chapingo,
9 Laboratorio Nacional de Investigación y Servicio Agroalimentario y Forestal, Texcoco,
10 56230, Estado de México, Mexico; S. G. Leyva-Mir, Universidad Autónoma Chapingo,
11 Departamento de Parasitología Agrícola, Texcoco, 56230, Estado de México, Mexico; G.
12 Márquez-Licona, Instituto Politécnico Nacional, Centro de Desarrollo de Productos
13 Bióticos, 62731, Yautepec, Morelos, Mexico
14
15 Lemon verbena (Aloysia citrodora; Verbenaceae) is an aromatic plant native to South
16 America. It is commercially planted in Mexico and widely used for medicinal and aromatic
17 purposes. During April of 2019, symptoms of powdery mildew were observed on 2000 lemon
18 verbena plants in an indoor nursery (19º30´52.9”N, 98º51´3.3”W) located in Texcoco, Estado
19 de México, Mexico. Disease incidence was 95 % and defoliation was observed in 35 % of
20 the plants. Signs of the pathogen were white powdery masses mainly on the upper leaf
21 surface. Symptoms included yellowing, necrosis, and early defoliation of the plants.
22 Morphological characters of the pathogen were examined microscopically considering
23 samples from 20 plants. Hyphae were hyaline, smooth, septate, flexuous, branched at right
24 angles, 3.3–4.0 mm wide. Hyphal appressoria were well developed, nipple-shaped to
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25 multilobed, solitary but mainly opposite in pairs. Conidiophores (n=30) were straight and
26 short, with foot cells cylindrical, straight to curved widening upwards and narrowed at the
27 base, 23.1–36.2 × 10.7–11.9 μm, followed by 1–3 shorter straight cells. Conidia (n=100)
28 were produced in chains, ellipsoid-ovoid to doliiform, 24.3–37.1 × 13.9–18.0 μm, without
29 fibrosin bodies, and germtubes developing multilobed appressoria in apical to subapical
30 position. Chasmothecia were not found. Morphological characters of the fungus correspond
31 with the previous descriptions of Neoerysiphe aloysiae by Wolcan and Sato (2007) and
32 Takamatsu et al. (2008). A voucher specimen was deposited in the Herbarium of the
33 Department of Agricultural Parasitology at the Chapingo Autonomous University under
34 accession no. UACH418. To confirm the identification, the internal transcribed spacer (ITS)
35 region and part of the 28S gene were amplified by PCR and sequenced. The ITS region of
36 rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). For amplification of
37 the 28S rRNA partial gene, a nested PCR was performed using the primer sets PM3
38 (Takamatsu and Kano 2001)/TW14 and NL1/TW14 (Mori et al. 2000) for the first and second
39 reactions, respectively. The concatenated sequence of both fragments was deposited in
40 GenBank under accession number MN999483. Phylogenetic analyzes using the maximum
41 likelihood method and including published ITS and 28S dataset were performed. The analysis
42 revealed that the concatenated sequence from the isolate UACH418 was grouped into a clade
43 with Neoerysiphe aloysiae with high posterior probability (99). Pathogenicity was
44 demonstrated by gently pressing diseases leaves against the surface of leaves of 10 healthy
45 plants. The same number of plants were left as controls. All plants were maintained in a
46 greenhouse at temperatures from 20 to 30ºC. All inoculated plants developed similar
47 symptoms to the original observations after 10 days, whereas the control plants remained
48 symptomless. The fungus on the inoculated plants was morphologically identical to that
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49 originally observed on diseased plants, thus fulfilling Koch’s postulates. The pathogenicity
50 test was repeated twice with identical results. Based on morphological and phylogenetic
51 analyses, the fungus was identified as Neoerysiphe aloysiae. This fungus has been reported
52 to cause powdery mildew on A. citrodora in Argentina by Wolcan and Sato (2007) and
53 Takamatsu et al. (2008). To our knowledge, this is the first report of N. aloysiae causing
54 powdery mildew on A. citrodora in Mexico. Based on our field observations, this disease is
55 a serious threat to the commercial plantations of lemon verbena in Mexico.
56
57 References:
58 Mori, Y., et al. 2000. Mycologia 92:74.
59 Takamatsu, S., and Kano, Y. 2001. Mycoscience 42:135.
60 Takamatsu, S., et al. 2008. Mycological Research 112:639-649.
61 White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications.
62 Academic Press, San Diego, CA.
63 Wolcan SM, Sato Y. 2007. Australasian Plant Disease Notes 2:113–114.
64
65
66 Funding: This research was financially supported by the Popular Autonomous University of
67 the State of Puebla (UPAEP), project UPAEP-1134.
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Powdery mildew of Aloysia citrodora caused by Neoerysiphe aloysiae. A. Leaves showing
white fungal growth. B. Initial symptoms of necrosis on the upper surface of a leaf. C.
Conidiophore arising from a hypha cell. D. Germinated conidium, conidial germ tube with
multilobed appressoria.
A
B
A
D
C
B
D
C
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Phylogenetic tree of Neoerysiphe aloysiae inferred from the combined dataset of the rDNA ITS
region and the 28S rDNA. The isolate UACH418 obtained from Aloysia citrodora in this study is
shown in bold. Maximum likelihood bootstrap support values are indicated at the nodes. Aspergillus
niger was used as outgroup.
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