Bol. R. Soc. Esp. Hist. Nat., 113, 2019: 67-69
First report of powdery mildew on Stevia rebaudiana (Bertoni) (Asteraceae) caused by Neoerysiphe cumminsiana (U. Braun) (Ascomycota) Primer reporte de mildiú polvoriento en Stevia rebaudiana (Bertoni) (Asteraceae) causada por Neoerysiphe cumminsiana (U. Braun) (Ascomycota)
Ricardo Carrasco1, Romina Ruiz-Arellano2, Luis Cedeño3, Kleyra Quintero3 & Gustavo Fermin4
1. Department of Biology, Faculty of Sciences, Universidad de Los Andes. Mérida, Venezuela. 2. Electronic Microscopy Center “Ernesto Palacios Prü”, Universidad de los Andes. Mérida, Venezuela . 3. Agricultural Research Institute, Faculty of Forestry and Environmental Sciences, Universidad de los Andes. Mérida, Venezuela. 4. Instituto Jardín Botánico de Mérida, Faculty of Sciences, Universidad de Los Andes, Mérida 5101. Mérida, Venezuela. [email protected]/[email protected]
Recibido: 8 de febrero de 2019. Aceptado: 16 julio de 2019. Publicado electrónicamente: 17 de julio de 2019.
Keywords: Asteraceae, Neoerysiphe cumminsiana (U. Braun), Stevia, Stevia rebaudiana (Bert.), Venezuela. Palabras clave: Asteraceae, Neoerysiphe cumminsiana (U. Braun), Stevia, Stevia rebaudiana (Bert.), Venezuela.
Abstract Neoerysiphe cumminsiana (U. Braun), the etiological agent of powdery mildew in diverse asteraceae plants, was recovered from symptomatic Stevia rebaudiana (Bertoni) plantlets grown under greenhouse conditions in Mérida, Venezuela. The etiology of the disease was confirmed by pathogenicity tests, and the identity of the fungus was performed using both morphologically and by sequencing and analysing the nuclear rRNA locus ITS4.
Resumen Neoerysiphe cumminsiana (U. Braun) causante de mildiu polvoriento en distintas Asteráceas fue observado en plantas de Stevia rebaudiana (Bertoni) obtenidas por cultivo in vitro, que se encontraban creciendo en condiciones de invernadero en el estado Mérida- Venezuela. El hongo en este hospedador crece ectofíticamente y aparece como un crecimiento blanco y polvoriento que lleva a la necrosis del tejido. Se realizaron pruebas de infección y patogenicidad que resultaron positivas y se confirmó su identidad por medio del análisis morfológico y de la comparación de secuencias del locus nuclear de ARNr conocido como ITS4.
1. Introduction Stevia rebaudiana (Bertoni) is a very economically important Asteraceae crop plant since it is widely used as sweetener, and as a raw material for the production of diverse biological active compounds (Takahashi et al. 2001; Chen et al. 2005; Ghanta et al. 2007; Jayaraman et al. 2008; Misra et al. 2011). The plant is commercially exploited
doi: 10.29077/bol/113/ce07_carrasco ISSN: 2659-2703 -67- R. Carrasco, R. Ruiz-Arellano, L. Cedeño, K. Quintero & G. Fermin
in many countries of the world; although most of the current production takes place in China, the main market is Japan (Brandle et al.2014). The plant shows increased susceptibility to fungal diseases when grown under conditions of excess water or in soils with high content of organic matter (Arturo et al. 2009). Other authors have reported the most important fungi causing various diseases in stevia plants, but not powdery mildew (Farr and Rossman, 2016). Neoerysiphe cumminsiana (U. Braun) is one of the five species belonging to the small fungal genus Neoerysiphe proposed by Braun (1999) in the order Erysiphales (Ascomycota). This phytopathogen has already been reported in the Americas (except the Caribbean), Asia (India, Israel and Japan), Ukraine and southern Africaas the etiological agent of powdery mildew on leaves and stems of plants belonging to more than 20 different genera of Asteraceae (Farr and Rossman, 2016). Here, we report the association of powdery mildew with stevia, caused by Neoerysiphe cumminsiana which was found in S. rebaudiana plantlets in Mérida state, Venezuela.
2. Materials and Methods In plants obtained by in vitro tissue culture, and later grown under greenhouse conditions in a commercial Figure 1A, Symptoms of the disease caused by Neoerysiphe cumm- facility, symptoms and signs of powdery insiana (U. Braun) in plants of Stevia rebaudiana (Bertoni); 1B, mildew and the presence of its putative Results from the pathogenicity tests in healthy leaflets ofStevia etiological agent were observed. Plants rebaudiana (Bertoni) inoculated with Neoerysiphe cumminsiana were referred to us for identification of (U. Braun); 1C,conidiophores; 1D, and conidia of the fungus the mildew fungus for which batches of isolated during the same assays. twelve healthy plants were used. Pathogenicity tests were performed by inoculating healthy leaves of stevia by direct contact with infected leaves. The inoculated plants incubated in laboratory with average temperature are 22ºC. Experiments were performed by serial duplicates using six plants each plus a mock control. Infected plants were observed daily, and the fungus analyzed morphologically as explained below. The fungal morphology was examined using mountings performed with transparent adhesive tape, using KOH 4% (pH 3.5) and acidlacto fuchsin 0.025%lacto fuchsin acid 0.025%, to improve the coloring penetration and to find the presence or absence of fibrosine corpuscules. Morphometric features and images were registered using and registered usingan Axioplan Zeiss microscope coupled with to a Canon A640 digital camera Molecular identification of the fungus was attained using a mixture of mycelium and conidia taken off symptomatic plants that were subsequently used to isolate DNA; PCR amplification of the rRNA nuclear locus was performed according to White et al.(1990) using primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). Reaction mixture consisted of 1 μl of total DNA (≥10ηg/µl), 0,5 µl of each primer (≈100µM), 5 μl of GoTaq® Green Master Mix (Promega, Madison, MI) and enough nuclease-free water to reach a final volume of 25 μl. PCR amplification was performed in an Applied Biosystems 2720 thermocycler using the following amplification profile: initial denaturation at 94°C for 120 sec, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 53°C for 60 secand extension at 72°C for 90 seconds, after which a final extension step at 72°C
-68- Comunicaciones especializadas First report of Stevia rebaudiana (Bert.) in Neoerysiphe cumminsiana for 5 min was included. Amplicons were evaluated by horizontal electrophoresis in 0.8% agarose gels stained with ethidium bromide,and their sizes checked using a 1 kbDNA molecular ladder (Promega).
3. Results and Discussion On stevia, the fungus grows ectophytically as a dense, white mycelium with a pulverulent aspect in both sides of the leaves, but preferentially on their adaxial sides (Figure 1A). Symptoms and signs developed further with the emergence of brownish necrotic spots resembling burns; leaflets became deformed, dry and brittle. Lesions progressed downwards, and the plant eventually died. The signs observed on the inoculated plants were similar to those observed in natural infections. Pathogenicity tests were positive as the same fungus could be recovered from induced lesions and symptoms resembled the ones observed before, which confirmed the fungus as the causal agent of the disease (Figure 1B). The fungus produced chains of conidia which lacked fibrosin bodies and in 100 of them the large and wide were measured; they are cylindrical with obtuse ends of 25.0- (29.4)-35.0 x 10.0-(13.4)-15.0 µm, and germination tube proceeds from their shoulders. The ratio length/width averaged 2.2, and appresoria were multilobed (Figure 1C and 1D). Amplicons of ca. 500 pb were obtained and sequenced after which sequence data were deposited at the GenBank public database under the accession number KX806655. The resulting sequence was compared to all available sequences at the same database through a BLAST search (Zhang et al. 2000), along with a similar comparison at the digital Mycobank server (www.mycobank.org). In both cases the sequence was more than 99% similar to diverse reported sequences of N. cumminsiana. Based on molecular data, as well as on the morphological analysis of the fungus, we conclude that the etiological agent of the powdery mildew on stevia plants observed under the described conditions is N. cumminsiana, as also demonstrated by fulfilling pathogenicity tests. This result not only widens the spectrum of countries where the fungus can be found, as well as the host range of the pathogen, but it also serves for the purpose of making stevia growers aware of another fungus able to challenge production, especially for low income, small holders who rely on the plant as a cash crop.
References Arturo, M.C., Torres-González, C., Peña, E.J., Díaz, J.E. 2009. Microorganismos patógenos de Steviare baudiana Bertoni. Bioagro, 21:173-178. Brandle, J,. Starratt, A., Gijzen, M. 2014. Stevie rebaudiana: its agriculture, biological, and chemical properties. Canadian Journal of Plant Science, 186: 527-536. doi: 10.4141/P97-114. Braun, U. 1999. Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series. 11:1-707. Chen ,T-H., Chen, S-C., Chan, P., Chu, Y-L., Yang, H-Y,, Cheng, J-T. 2005. Mechanism of the hypoglycemic effect of stevioside, a glycoside of Stevia rebaudiana. Planta Medica, 2:108-113. doi: 10.1055/s-2005-837775. Farr, D.F., Rossman, A.Y. 2015. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA.
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