Occurrence of Pyricularia oryzae Triticum in plants of the genus Urochloa in Brazil

ABSTRACT: In this study Pyricularia spp., P. oryzae and the P. oryzae pathotype Triticum (PoT) were detected and identified in leaf segments of forage and invasive grasses located in or next to wheat fields. In 2018 and 2019, 66 samples of lesion leaf segments of Urochloa and other grasses were collected in Londrina (PR), Patos de Minas (MG), and Uberaba (MG). The detection and/or identification of the pathogens on the samples was conducted using moist chamber procedures and with the primers MoT3 and Pot2 by PCR. There were DNA amplification with the primer MoT3 (specific for PoT) for 13 (19.69%) of the samples, all of them from Urochloa. The finding that Urochloa hosts PoT at a relatively high rate raises concerns about the importance which these plants may have on the wheat blast cycle as an alternative host for the pathogen and/or source of inoculum for the disease.

The occurrence of the wheat blast is more common in the tropic regions where the yield losses caused by this disease may reach 100% (MACIEL, 2018) and its presence is especially associated with wheat head blast (CRUZ & VALENT, 2017).The condition of P. oryzae with a wide range of hosts including forage and weeds plants (MACIEL et al., 2014;TOSA & CHUMA, 2014;CRUZ & VALENT, 2017) raises concerns about the role and importance that these hosts play in the wheat blast cycle, both as a source of primary inoculum and as part of the process of generating genetic variability in this species of fungus (CASTROAGUDÍN et al., 2016).
In Brazil, plants of the genus Urochloa (syn.Brachiaria) have received greater attention regarding their possible interference in the wheat blast cycle because they are widely disseminated forages in the Brazilian agricultural system (CASTROAGUDÍN
To know more about the role of alternative hosts in the wheat blast cycle is important to confirm if PoT is really infecting these hosts in the natural environmental, especially in the wheat fields.This can be accomplished by accurate methods of diagnosis and identification of the pathogen.Conventionally used, the cultural diagnosis of P. oryzae based on "the moist chamber procedures" requires a minimum incubation period for the fungus (KRUG, 2004) and the use of a microscope to find the reproductive structures of the pathogen.The diagnostic technique using molecular methods based on the polymerase chain reaction (PCR) in plant tissues is a precise, fast, and sensitive option for several pathosystems (LAU & BOTELLA, 2017).
Some PCR primers for the detection of P. oryzae infecting plants have been reported.Two of them are specific to PoT, MoT3 (PiECK et al. 2017) andC17 (THIERRY et al., 2020), which amplify DNA fragments with 361 and 500 base pairs (bp), respectively.In addition to these, the PCR primer Pot2 has also been used; although, it is not specific for any pathotype of P. oryzae, that is, it amplifies 410 bp fragments in any positive detection of P. oryzae (KACHROO et al., 1994).
The objective of this study was to detect and identify Pyricularia spp., P. oryzae, and its pathotype PoT in leaf segments of forage and invasive grasses located into or next to wheat fields Leaf segments with symptoms of lesions were collected from forage and invasive grass plants in agricultural areas of three Brazilian municipalities: Londrina, PR; Patos de Minas, MG; and Uberaba, MG.These samples were sent to Embrapa Trigo, Passo Fundo, RS, where, in the Phytopathology and Biotechnology laboratories, they were subjected to evaluation for detection and identification of P. oryzae and PoT.Initially, the samples were registered and subjected to drying at 40-50 ºC for 30 min, and stored at -20 ºC.From each sample and using a scissors, two very homogeneous sub-samples were formed in relation to the symptoms they presented; A-sub-samples and B-sub-samples.Depending on the sub-sample, A or B, each sub-sample was subjected to one of the following methods of detection and identification of plant pathogen fungi; (A) cultural, subjected to moist chamber procedures, and (B) molecular, by PCR technique.
Moist chambers were conducted in Petri dishes.Three sheets of blotting paper were placed inside each plate and moistened with distilled and autoclaved water.Two leaf segments of each A-subsample were allocated per plate, in four replications.The plates were randomly placed under continuous fluorescent illumination (25 μm m 2-1 s -1 ) at 25 ºC +/-2, and photoperiod of 12 h, for 120 h.After 7 days, the leaf segments were carefully examined with the aid of stereoscopic magnifiers and compound light microscopes.Positive detections of Pyricularia spp.were confirmed based on the visualization of conidiophores and conidia of the fungus.
The molecular detection and identification of P. oryzae and PoT was carried out in the DNA extracted from the B-sub-samples.Each one of them provided material to conduct two DNA extractions and each DNA extraction was done in five lesioned leaf segments of approximately 2 x 2 cm.The PCR primers used were MoT3 (PiECK et al., 2017) and Pot2 (KACHROO et al., 1994).DNA amplification was done in a capillary electrophoresis system.Data were evaluated using the GENEMAPPER ® iD software -Version 3.7, generating the electropherograms.
Cultural detection via moist chamber allowed us to identify reproductive structures of Pyricularia spp. in 32 of the 66 sub-samples evaluated, equivalent to 48.48% of the sub-samples (Table 1).From the 2018 and 2019 sub-samples, out of 32 and 34, 14 and 18 of them showed sporulation of Pyricularia spp., respectively.These results represent;respectively, 43.75% and 52.94% of the subsamples with positive detection for Pyricularia.There was abundant production of conidia in all samples with positive detection of the fungus by the cultural method.Pyricularia sporulation in symptomatic lesions of plants of the genus Urochloa occurred in typical and characteristic blast lesions, with elliptical shape and gray center.All samples that showed conidia sporulation of Pyricularia spp. in the cultural diagnosis also had positive detection when PCR primer Pot2 was used.Out of 66 samples, in 23 there was amplification for the Pot2 marker (34.85%), and in 13 for the MoT3 marker (19.69%).From the 2018 sub-samples, out of 32, 15 amplified for Pot2 (46.87%) and 7 for MoT3 (21.87%).From the 2019 sub-samples, out of 34, 18 amplified for Pot2 (52.94%) and 6 for MoT3 (17.65%).The positive control resulted in PCR amplification for the two primers used in the study.The DNA used for that was from samples of wheat plants, which had been subjected to artificial inoculation with PoT conidia.The results obtained demonstrated PCR efficiency and sensitivity, as amplification was observed in all wheat samples, except for the negative control (without DNA) and in one sample in which was used DNA of Fusarium graminearum.It was also detected the presence of P. oryzae in two forage species not belonging to the genus Urochloa, which were the following ones, Cynodum plectostachyus and Melinis minutiflora.
As the identification of PoT in the subsamples was based on the DNA amplification promoted by the PCR primer MoT3, it is important to consider some observations already registered regarding the specificity of this diagnostic system for the causal agent of wheat blast.PiECK et al. (2017) testing PCR primer MoT3 observed no amplification for a wheat-infecting isolate and presence of amplification for a grass weed isolate (Bromus tectorum) collected into a wheat field.GUPTA et al., 2019 also verified that MoT3 did not distinguish between rice and wheat isolates from Bangladesh.According to the authors of the MoT3 diagnostic primer, different PCR conditions, reagents, and thermocycler machines could be the reasons for the unexpected results.After that, the authors retested the specificity of the MoT3 using the same isolates and it was reconfirmed the previous results (YASUHARA-BELL et al., 2019).
The finding about the occurrence of PoT in Urochloa raises a lot of concern about wheat blast in Brazil, especially in places where this disease occurs more frequently.These concerns refer to potential consequences of this biological event.One of them may be the possibility of increasing of the genetic variability of the pathogen under natural conditions.This possibility has relation with the records done by Urashima et al (1993) about the occurrence of sexual recombination between isolates of PoT and of P. oryzae from Urochloa plantaginea.This situation may mean greater difficulties for the grower, since new variants of the pathogen, eventually, may present (a) ability to break any resistance to wheat blast present in wheat cultivars; or (b) have less sensitivity to fungicides used to control this disease.It is also important to consider the particularities of the wheat crop in Brazil, where the fields are established in sequence with summer crops (soybean and corn), providing the return of wheat plants to the field after a period that normally lasts from 5 to 8 months.As PoT does not survive in wheat straw for more than 5 months (PiZOLOTTO et al., 2019), plants such as those of the genus Urochloa can play very important key role in the wheat blast cycle as an intermediate host for the pathogen and source of inoculum for the disease.
The findings that Urochloa plants host PoT at relatively high rate raises concerns about their importance on wheat blast cycle and based on that we recommend further studies on the subject.Concerns come from the condition that Urochloa is widely used as forage in Brazil, normally established in locations relatively close to wheat fields, and some plant species of this genus are weeds on wheat crop.

ABSTRACT:
In this study Pyricularia spp., P. oryzae and the P. oryzae pathotype Triticum (PoT) were detected and identified in leaf segments of forage and invasive grasses located in or next to wheat fields.In 2018 and 2019, 66 samples of lesion leaf segments of Urochloa and other grasses were collected in Londrina (PR), Patos de Minas (MG), and Uberaba (MG).The detection and/or identification of the pathogens on the samples was conducted using moist chamber procedures and with the primers MoT3 and Pot2 by PCR.There were DNA amplification with the primer MoT3 (specific for PoT) for 13 (19.69%) of the samples, all of them from Urochloa.The finding that Urochloa hosts PoT at a relatively high rate raises concerns about the importance which these plants may have on the wheat blast cycle as an alternative host for the pathogen and/or source of inoculum for the disease.Key words: wheat blast, primer MoT3, source of inoculum.RESUMO: Neste estudo Pyricularia spp., P. oryzae e o patótipo Triticum (PoT) de P. oryzae foram detectados e identificados em segmentos foliares de forrageiras e gramíneas invasoras de lavouras de trigo.Em 2018 e 2019, foram coletadas 66 amostras de segmentos foliares lesionados de Urochloa e outras gramíneas em Londrina (PR), Patos de Minas (MG) e Uberaba (MG).A detecção e/ou identificação dos patógenos nas amostras foi realizada por meio de procedimentos de câmara úmida e com os iniciadoresMoT3 e Pot2 por PCR.Houve amplificações de DNA com o primer MoT3 (específico para PoT) em 13 (19,69%) das amostras, todas provenientes da Urochloa.O resultado de que Urochloa hospeda PoT em uma taxa relativamente alta levanta preocupações sobre a importância que essas plantas podem ter no ciclo de brusone do trigo como hospedeiro intermediário para o patógeno e / ou fonte de inóculo para a doença.Palavras-chave: brusone do trigo, primer de PCR MoT3, fonte de inóculo.

Table 1 -
Detection and identification of Pyricularia spp., P. oryzae and P. oryzae Triticum on symptomatic segments of poaceae plants collected in three Brazilian municipalities.