Eur J Plant Pathol https://doi.org/10.1007/s10658-018-01645-3 Occurrence of the complete cycle of Puccinia sorghi Schw. in Argentina and implications on the common corn rust epidemiology F. A. Guerra & R. L. De Rossi & E. Brücher & E. Vuletic & M. C. Plazas & G. D. Guerra & D. A. Ducasse Accepted: 19 November 2018 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2018 Abstract The life-cycle of Puccinia sorghi, a heteroe- on O. conhorriza in Córdoba may play an important role cious fungus, consists of five well-defined spore stages. in generating new variants of P. s org hi in Argentina, The uredinial and telial stages are completed on the allowing a constant adaptation of the pathogen to the primary host (maize) whereas spermagonial and aecial environment of the different corn production zones. stages occur on Oxalis spp., a perennial and widespread weed. Portions of corn leaves with telia were surface Keywords Common rust . Maize . Corn . Oxalis . Zea sterilized and placed in Petri dishes with 2% water agar mays L. and maintained in a growth chamber at 25 ± 1 °C and photoperiod of 16 h light and 8 h dark for 48 h to induce the formation of basidia and basidiospores. Oxalis Introduction conorrhiza plants were inoculated with those basidio- spores, to confirm the generation of spermagonia with Common corn rust, caused by Puccinia sorghi Schw. spermatia, and subsequently aecia with aeciospores. (P. s org hi), is one of the most important leaf diseases in Corn plants were then inoculated with aeciospores to the corn cropping area of Argentina (INTA 1980; confirm the formation of urediospores and teliospores. Gonzalez 2005). It is a very common disease, appearing The aecial phase of common corn rust was confirmed to every year with different levels of incidence and severity occur on O. conhorriza and the descriptions of spore in the corn producing areas of Argentina (Gonzalez stages in Argentina are now reported in this work, 2005). The causal agent is P. sorghi, a biotrophic, confirming a potential sexual source of variability of heterocius and macrocyclic fungus, belonging to the P. s org hi. The natural occurrence of aecial infections Basidiomycota group. The life cycle involves corn (Zea mays L.) as the telial host and several Oxalis species as aecial hosts. The complete cycle of P. s org hi F. A. Guerra : R. L. De Rossi : E. Brücher : M. C. Plazas : G. D. Guerra : D. A. Ducasse includes the generation of five spores stages: telio- Universidad Católica de Córdoba (U.C.C.), Córdoba, Argentina spores, basidiospores, spermatia, aeciospores and ure- diospores (Biswanath 2016). * : : F. A. Guerra ( ) E. Brücher E. Vuletic Teliospores, basidia and basidiospores are the main Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Córdoba, Argentina phases in rust life cycles for overwintering and they are e-mail: [email protected] responsible for the formation of new physiologic races (Anikster and Wahl 1979). Some teliospores are consti- * D. A. Ducasse ( ) tutively dormant, which is often a mechanism to protect Instituto de Patología Vegtal (IPAVE-CIAP) Instituto Nacional de Tecnología Agropecuaria, Córdoba, Argentina the fungus during unfavourable environmental period e-mail: [email protected] (Anikster 1986) and require processes for germination Eur J Plant Pathol of the teliospore. These processes can be activated by: 64°09’33.1^ W), telia from corn leaves were collected storage outside, different humid and temperature range and stored in a herbarium. After 5 months, portions of conditions, alternation of wet/dry conditions of the those leaves with telia were surface sterilized with 2% spore, exposure to different light regimes, and/or inter- sodium hypochlorite for three minutes, rinsed three nal factors (Mendgen 1984). times with sterile water for two minutes and placed in In some areas of the world, P. sorghi teliospores Petri dishes with 2% water agar. Plates were put into a germinate (usually in the spring) to generate basidia growth chamber at 25 ± 1 °C and photoperiod of 16 h and basidiospores. The latter germinate and penetrate light and 8 h dark, and the germination was monitored only into Oxalis spp. leaves (Pataky 2016). In most every 12 h for production of basidia with basidiospores. temperate areas of the world, P. s org hi does not infect Oxalis spp. after surviving the winter; hence, aeciopores Inoculation of O. conorrhiza plants are not the primary inoculum of corn rust. Initial infec- tion of corn coincides with the arrival of air-borne Seeds of O. conorrhiza were sowed in 850 ml plastic urediospores from nearby tropical and subtropical re- pots containing a substrate composed of 70% fertile soil gions, where the aecial stage appear on Oxalis spp. In and 30% vermiculite. Plants were inoculated at the 20 other areas, the pathogen survives on volunteer maize leaflet stage and after being kept for 24 h. in a saturated plants and urediospores are dispersed by wind until they humidity environment. reach susceptible corn plants, infecting them and devel- Portions of corn leaves with telia were disinfected oping the uredinal stage continuously (Reis et al. 2004). and placed in Petri dishes with 2% water agar for 24 h Ten Oxalis species have been reported as hosts of with the aforementioned methodology. P. s org hi over the world (Farr and Rossman 2016). In Two inoculation methodologies were applied; one Argentina Godoy and Bruni (1952)reportedP. s org hi (treatment A) simulating infected stubble on the soil, on O. corniculata in the Buenos Aires province at an and the other (treatment B) simulating inoculum from urban environment, but not in rural fields or near corn unharvested corn stalks. In treatment A, leaf portions crops fields. In Córdoba province, Guerra et al. (2016) with basidiospores were removed from the plates and reported natural infections of P. s org hi on O. conorrhiza placed on the soil surface of a pots where Oxalis plants in different rural locations near corn crops. Among the were growing and covered with transparent plastic bags alternate hosts of P. s org hi, O. conorrhiza is a South to maintain a saturated environment for 48 h. In treat- American native perennial specie distributed throughout ment B, the methodology described by Morin et al. Argentina, Brazil, Paraguay and Uruguay (Belgrano (1993) was applied as follows: a transparent plastic tube et al. 2008). Its geographical distribution coincides with was placed over the pots where Oxalis plants were the greatest corn production areas of Argentina growing and an inverted plate containing leaves with (MAGyP 2016). Although the aecial stage of P. s org hi basidiospores was placed on top, and was kept closed has been previously reported in Argentina (Godoy and for 48 h. The control treatment consisted of the same Bruni 1952;Guerraetal.2016), the generation in nature procedures but without inoculum. The procedures were of all five spores stages, including basidiospores and repeated four times each. Inoculated Oxalis pots were determination of the environmental conditions required kept in a plant growth chamber at 25 ± 1 °C, with 70 ± for the development of the complete cycle, have not 10% relative humidity and a photoperiod of 16 h of light been documented. Here we present evidence that all five and8hofdarkness. stages of P. s org hi occur in the field in Argentina. Inoculation of corn plants Materials and methods Seeds of a P. s org hi susceptible hybrid corn (experimen- tal hybrid, KWS Argentina S.A.) were sowed in 850 ml. Collection and conditioning of samples and germination plastic pots, containing a substrate composed of 70% of basidia fertile soil and 30% vermiculite. When plants reached the three fully expanded leaf stage, they were placed for At the end of the 2015–16 corn crop campaign on a field 24 h in a saturated humidity environment. Aeciospores located in Córdoba province (31°10′04.1B S were manually extracted from mature aecia on Eur J Plant Pathol O. conorrhiza infected leaves and placed in a 1.5 ml eppendorf tube with 1 ml of sterile water. After shaking, the suspension of aeciospores was sprinkled on the seedlings. Inoculated corn were kept in a humid saturat- ed environment for another 24 h and then, the pots were placed in a plant growth chamber at 23 ± 1 °C, with 70 ± 10% relative humidity and a photoperiod of 16 h light and8hdark. Microscopy, morphometry and image capturing For morphometric evaluation and image capturing, the following methodologies were carried out: i) extraction of produced spores (urediospores, teliospores, aeciopores and basidiospores); and ii) thin sections of the fruiting bodies (aecia and spermagonia). Uredio- Fig. 1 Teliosporas of P. so rgh i with probasidia spores, teliospores and aeciospores, collected from por- tions of symptomatic tissue from leaves of each host, were placed in a 1.5 ml tube with 0.5 ml of sterile water μ and shaken for 60 s. Subsequently, 10 l of the suspen- telia initially presented as flat, opaque, compact, and sion were placed on a microscope slide and observed dry-looking, and at the time of germination they looked under the microscope. Alternatively using a stereoscop- bright, moist, voluminous and lax. ic microscope and a histological needle, structures de- Mendgen (1984) reported that teliospores of P. s org hi veloped within the telia were collected and placed on a present an intermediate maturity, and thus dormancy slide. A Zeiss Primo Star optical microscope was uti- length is variable allowing them to germinate lized, and photographs were taken using a Canon Power immediately or only after a few months after apearing. Shot SX 30 IS camera. The morphometric parameters Pavgi (1975) stated that termination of the dikaryotic for the pathogen characterization were determined and condition is essential for germination.