Pythium arrhenomanes causal agent of root rot on yellow maize in Mexico

Maize (Zea mays) is one of the most important crops in Mexico. During November of 2015, symptoms of wilting were observed on the maize plants variety Overland (Syngenta) located in Copandaro, Michoacan, Mexico (19° 53′ 37” N, 101° 14′ 29.4” W) with 60% incidence in some areas. The disease affected mature plants at reproductive stage, chlorosis on leafs was initially observed and then plants wilted and died (Fig. 1). The crown and the main root showed dark brown necrosis, meanwhile the adventitious roots were pale brown. Transversal tissue sections showed abundant coenocytic mycelia. The objective of this work was to identify the causal agent of wilt and root rot on maize plants in Michoacan.

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Samples of diseased plants were collected and taken to the laboratory. The isolation of the pathogen was performed cutting small transversal sections of the crown, approximately of 0.5 cm² with pathogen symptoms, that were disinfested with a solution of 10% commercial chlorine (Clorox^®) during 30 s, rinsed with distilled sterile water, dried with sterile filter paper, placed on potato dextrose agar (PDA) media, and incubated at 24 °C for 24 h. When mycelial growth was observed isolate purification was performed, each mycelium was transferred on water agar for purification and each grown hyphal tip were further transferred on PDA. The isolate was transferred to cryovials with distilled water and kept at 15 °C with the code CPV-669. The isolate was identified in terms of morphological characterization and molecular phylogenetics, followed by pathogenicity tests.

Sporulation and gametangial formation was induced placing PDA disks with mycelia in an empty Petri dish with autoclaved grass blades, they were incubated at 26 °C for 48 h (Kerns and Tredway, 2010). Coenocytic mycelia, lobated sporangia (Fig. 2b), oogonia with antheridia (Fig. 2c) and plerotic oospore were observed (Fig. 2d). The diameter of oospores was in the range 31 to 45 μm and the number of antheridia per oogonia was 3 to 17. These characteristics match the ones reported for Pythium arrhenomanes by Chen and Hoy (1993) and Van Der Plaats-Niterink (1981). Antheridia in the sexual phase were only observed once in the cultures, this coincides with the description of P. arrhenomanes reported by (Kerns and Tredway, 2010).

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For molecular characterization genomic DNA was extracted (Saghai-Maroof et al. 1984) and the 28S rDNA region was amplified using the primers ITS4 and ITS5 (White et al. 1990). Subsequently the cytochrome oxidase (COXII) gene was amplified with the primers FM35 (Martin 2000) and FMPHY-10b (Martin et al. 2004). Similarity of the sequence on ITS region was 100% with that of P. arrhenomanes strain BR605 from Canada (Genbank Accession No. HQ643468.1), and similarity on COXII was 99% with P. arrhenomanes strain ATCC96526 from sugarcane in Louisiana USA (Genbank Accession No. AB095053.1). The sequences obtained were deposited in NCBI for ITS as Accession No. KY570311 and for COXII as KY581583 (strain CPV-669). ITS and COXII concatenated aligned sequences were analyzed under maximum parsimony (MP), to infer phylogenetic relationships with the program PAUP*4.0a157 (Swofford 2002). A phylogenetic analysis including sequences of the species: P. graminicola, P. aristosporum, P. arrhenomanes and P. aphanidermatum (Kageyama et al. 2005), generated a tree that was visualized with Mesquite3.31 (Maddison and Maddison 2017). Our strain CPV-669 was grouped with the strain ATCC96526 which confirmed the results of the alignment analysis (Fig. 3).

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Pathogenicity tests were performed with maize seedlings of the cultivar Overland. Seeds were washed three times with sterile distilled water for 10 min to remove fungicides and insecticides. Seeds were germinated in Falcon tubes (50 mL) with 27 g of the substrate Sunshine Mix #3. Prior to use substrate was sterilized at 120 °C/15 lb. for 30 min. The tubes with the seeds were placed in a growth chamber at 27 °C with a photoperiod of 12/12 h (day/night) and watered with 10 mL every 72 h. The inoculum was prepared by pre-inoculating axenic cucumber tissue (3 mm thick and 9 mm in diameter) with P. arrhenomanes from a four day old PDA mycelial plug, that was incubated at 26 °C for 24 h. Fourteen days after seed germination one cucumber disk with mycelia of P. arrhenomanes was placed on the base of the stem of twenty individual plants and covered with substrate. Other twenty control plants were inoculated with PDA discs without P. arrhenomanes. Five days after inoculation the plants started to show symptoms of reduction of leaf length and plant height, (Fig. 2 a). Ten days after inoculation the pathogen was reisolated from diseased root tissue. The isolates showed the same morphological characteristics as P. arrhenomanes. The isolated was deposited in the National Collection of Genetic Resources of Mexico under the registry number: CM-CNRG 5416.

According to the morphological and molecular characteristics the isolate was identified as P. arrhenomanes. This pathogen was reported for the first time causing symptoms of root rot on corn in the District of Columbia, Canada by Drechsler (1928). P. arrhenomanes is pathogenic on different cereal crops including wheat and rice (Vanterpool 1930; Van Buyten et al. 2013). In Mexico this oomycete has only been reported pathogenic on sugar cane (Riess and Flores 1976; Fernández-Pavía et al. 2015). To the best of our knowledge this is the first report of P. arrhenomanes causing wilting, root necrosis and death of maize plants in Mexico.