Pyricularia pennisetigena and P. zingibericola from invasive grasses infect signal grass, barley and wheat 1

Fungal species from the Pyricularia genus are associated with blast disease in plants from the Poaceae family, causing losses in economically important crops such as rice, oat, rye, barley, wheat and triticale. This study aimed at characterizing the pathogenicity spectrum of P. pennisetigena and P. zingibericola to signal grass, barley and wheat, as well as comparing them with those from the species P. grisea and P. oryzae pathotype Triticum , which occur widely in the Brazilian agroecosystem. Twenty isolates of Pyricularia spp. were obtained from infected leaf samples of invasive plant species from wheat fields. The isolates classification into distinct Pyricularia species was done using molecular phylogeny based on actin and calmodulin genes. Pyricularia pennisetigena and P. zingibericola inoculated on plant leaves, at a concentration adjusted to 10 5 conidia mL -1 , were pathogenic to signal grass, barley and wheat, with varying levels of aggressiveness.


INTRODUCTION
The Pyricularia genus includes several pathogenic fungi species that are associated with blast disease in more than 50 Poaceae plants (Ou 1985).Among these species, Pyricularia oryzae is the most important pathogen, due to its global distribution on plants from the Poaceae family.The blast disease caused by P. oryzae is responsible for severe losses in crops of high economic importance such as rice (Oryza sativa), oat (Avena sativa), signal grass (Urochloa spp.), rye (Secale cereal), barley (Hordeum vulgare), maize (Zea mays) and wheat (Triticum aestivum) (Bailey & Eijnatten 1961, Igarashi et al. 1986, Urashima et al. 1993, Anjos et ABSTRACT RESUMO al. 1996, Couch & Kohn 2002, Goulart et al. 2003, Couch et al. 2005, Marchi et al. 2005).
The adoption of the Pyricularia oryzae designation as the species associated with blast disease is recent.Until the early 2000s, Pyricularia grisea was considered the pathogen of blast disease on rice, signal grass, barley and wheat (Urashima et al. 1993, Marchi et al. 2005).A molecular phylogenetic reclassification proposed by Couch & Kohn (2002) showed that P. grisea was exclusively associated with blast disease on grasses from the Digitaria genus, whereas P. oryzae was associated with rice, wheat and other cultivated grasses or invasive plants (e.g.goosegrass).Based on this study, a new international classification became necessary.
The Triticum pathotype of P. oryzae is associated with wheat blast in South America, especially in Brazil (Tosa & Chuma 2014).Since it was first reported in 1985, in the Paraná State (Igarashi et al. 1986, Goulart et al. 2007), blast disease on wheat ears has become a very important disease, causing crop losses of 40-100 % (Goulart & Paiva 2000, Torres et al. 2009).Due to the lack of varieties with high levels of genetic resistance and efficient chemical control to manage the disease, the pathogen has become widely distributed in all wheat growing fields in Brazil (Maciel et al. 2013, Castroagudín et al. 2014).This disease has also been detected in wheat fields in northern Argentina, Bolivia and Paraguay (Duveiller et al. 2010), but it had never been reported outside South America until 2016.Recently, wheat blast disease was considered one of the most important quarantine diseases and a threat to the wheat crop in the USA (Duveiller et al. 2010) and in Bangladesh (Callaway 2016, Malaker et al. 2016).
In the survey of invasive Poaceae species infected with blast disease described in this study, carried out in wheat fields of the Mato Grosso do Sul and Paraná States, during the 2012 and 2013 growing seasons, an association of P. pennisetigena and P. zingibericola with several of the grass species sampled, besides P. grisea and P. oryzae, was detected.Despite the recent report in the Brazilian agroecosystem (Klaubauf et al. 2014), the occurrence of the P. pennisetigena and P. zingibericola species could be more common and older than assumed.However, the potential of P. pennisetigena and P. zingibericola as pathogens to economically important Poaceae hosts in the Brazilian agriculture is still unknown.
Therefore, this study aimed at characterizing the pathogenicity spectrum of P. pennisetigena and P. zingibericola to signal grass, barley and wheat, as well as comparing them with those from the P. grisea and P. oryzae species, which occur widely in the Brazilian agroecosystem.The role of invasive plants as possible additional hosts of P. oryzae on wheat was also described.

MATERIAL AND METHODS
For the comparative study of the pathogenicity spectrum of Pyricularia species, 20 isolates were obtained from leaves or ears of invasive plants sampled from wheat fields in South-Central Brazil (8 isolates of P. pennisetigena; 3 isolates of P. zingibericola; 5 isolates of P. oryzae from invasive plants and 1 from wheat; and 3 isolates of P. grisea) (Table 1).The samplings of diseased plants were conducted in wheat fields in the Mato Grosso do Sul (MS), Minas Gerais (MG) and Paraná (PR) States and Brasília (DF), between May and August, during the 2012 and 2013 growing seasons (Table 1).
For fungal genomic DNA extraction, the isolates were grown on potato dextrose broth containing streptomycin (0.050 g L -1 ) and chloramphenicol (0.050 g L -1 ), and incubated for 7 days at 25 o C, with constant stirring at 75 rpm.The mycelium obtained from each isolate was filtered, frozen at -20 o C and lyophilized for 24 h.The genomic DNA extraction from the fungal mycelium was performed using a Genelute Plant Genomic DNA Mini Kit (Sigma-Aldrich, Brazil), following the manufacturer's instructions.
The amplification program used in the Mastercycler Nexus PCR thermocycler (Eppendorf, Hamburg, Germany) included initial denaturation at 95 ºC, for 5 min; followed by 35 cycles at 95 ºC, e-ISSN 1983-4063 -www.agro.ufg.br/pat-Pesq.Agropec.Trop., Goiânia, v. 46, n. 2, p. 206-214, Apr./Jun.2016 for 45 s; annealing at 58 ºC for the ACT gene and at 55 ºC for the CAL gene, for 1 min, with extension at 72 ºC, for 1 min; and a final extension at 72 ºC, for 8 min.The PCR products were sent for sequencing at the Macrogen Inc. company (Seoul, South Korea), which used the automated PE Applied Biosystems ABI-3730 sequencer.The sequences obtained were analyzed and aligned using the Geneious R 6.7.1 software (Biomatters, New Zealand).The experimental sequences of ACT and CAL genes obtained from the isolates in this study were submitted to the NCBI GenBank (Table 1).
Prior to the phylogenetic analysis, the JModelTest version 2.1.7software (Darriba et al. 2012) was used to determine the nucleotide substitution model that best fitted the data.The model adopted was K80 + invariable sites.The phylogenetic analysis was performed based on the actin and calmodulin gene sequences aligned and concatenated, based on the maximum likelihood method (ML), using the PhyML algorithm and heuristic searches (Guindon & Gascuel 2003) implemented in the Geneious R 6.7.1 software (Biomatters, New Zealand).The support for the internode branches of the maximum likelihood (ML) tree was tested by bootstrapping the data with 10,000 resamplings.
The isolates of Pyricularia spp.were grown on Petri dishes containing oatmeal agar medium and kept under photoperiods of 12 h and at a temperature of 25 ºC, for 5 days.Subsequently, 5 mm discs containing mycelium and conidia of Pyricularia spp.For inoculum preparation, the mycelium was scraped with the help of a sterile spatula and the fungal colonies were washed with distilled water plus the spreader-sticker Tween 80 (2 drops L -1 ).The concentration of conidial suspensions was determined in a Neubauer chamber, adjusting it to 10 5 conidia mL -1 for inoculation (Maciel et al. 2013).The conidial suspension volume for the inoculation of young plants was 50 mL, for a total of 15 pots (five for each host).
The pathogenicity spectrum of Pyricularia spp.isolates was determined by the inoculation on signal grass (U. brizantha) cv.Piatã, barley (H.vulgare) cv.BRS Korbel and wheat (T.aestivum) cv.Anahuac 75.These plants were sown in two seasons under screenhouse conditions, firstly on June 8 and secondly on June 18, in 2015.About 10 to 12 seeds were sown in 300 mL plastic pots containing Tropstrato HT Hortaliças plant substrate (Vida Verde, Campinas, SP).Prior to the inoculation, the plants were kept in a screenhouse at temperatures ranging 25-30 ºC, with a 12 h regimen of light and daily irrigation.A fertilization (2.6 g pot -1 ) with the N-P-K formula (10-10-10) was performed 10 days after sowing.
Two weeks after emergence, when the plants tested were at the phenological stage of four leaves, thinning was performed, so that only six plants per pot were left.Each experimental plot consisted of one pot with six plants, totaling 30 plants per host.The inoculation was performed subsequently.With the assistance of an air pressure pump spray, the conidial suspension was sprayed onto each plant, until the leaf surface (adaxial and abaxial) was completely wet.After inoculation, the plants were incubated for 24 h in the dark, in a phytotron chamber, at a temperature of 24 ºC, under nebulization.The experimental plots were then transferred to an acclimatized greenhouse with automatic control of air temperature (25 ºC) and relative humidity (90 %), and with 12 h of natural sunlight.The pots were irrigated daily.
The experimental design adopted for the three host species tested was completely randomized, with five replicates.For each host species, the experimental treatments were represented by 20 isolates and one check (negative control).This experiment was replicated once and conducted in two seasons.
The plants were assessed 7 days after inoculation.Five leaves with blast symptoms per pot were photographed using an Alpha 5000 Sony digital camera, with 16-50 mm interchangeable lens, attached to a monopod, at a fixed distance of 20 cm.The pathogenicity spectrum of the isolates and the levels of aggressiveness were determined based on the percentage of infected leaf area of plants with the symptoms of leaf blast.The infected leaf area was determined using the Assess Image Analysis Software for Plant Disease Quantification version 2.0 of the American Phytopathological Society (Lamari, Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada).
Analysis of variance was performed using the SAS software version 9.1 (SAS Institute Inc., Cary, North Carolina, US).The PROC MIXED procedure was used to analyze the treatment effect, and the Scott-Knott test (p < 0.05) was used for comparison between the groups of isolates.

RESULTS AND DISCUSSION
In general, this study resulted in the phylogenetic identification and in the elucidation of the pathogenicity spectrum of P. pennisetigena and P. zingibericola, identified by surveying fungi associated with blast disease in invasive plants from wheat fields in South-Central Brazil.
New taxonomic alterations have been recently proposed, such as the definition of the new Pyriculariaceae family, designation of new genera, introduction of new species and the allocation of species previously described in the Magnaporthe genus to the Pyricularia genus.The Magnaporthe genus, previously used to designate the sexual phases of P. grisea and P. oryzae (i.e.M. grisea and M. oryzae), has been less used.Furthermore, there was the inclusion of new species of Pyricularia previously not described as pathogens of Poaceae, such as P. pennisetigena and P. zingibericola (Klaubauf et al. 2014).
In the phylogenetic analysis, branch I, with 99.9 bootstrap support, grouped the isolates of Pyricularia oryzae obtained from invasive plants [13.0.033i (from Cynodon spp.), 13.0.017i(from Elionurus candidus), 12.0.029i(from Rhynchelytrum repens), 12.0.005(from undefined Poaceae) and the isolate 12.0.222(from Panicum maximum)], the isolate of P. oryzae from wheat (12.1.005)and two other isolates of P. oryzae, whose sequences were retrieved from the GenBank, with one being isolated from wheat (Triticum pathotype) and the other from rice (Oryza pathotype).
In fact, the latest study elucidating the phylogenetic relationship between several Pyricularia species has indicated that isolates obtained from rice and wheat were grouped into the clade P. oryzae (Klaubauf et al. 2014).These isolates, although specific to those hosts, potentially have a common evolutionary origin (Tosa & Chuma 2014).Our observations indicated that P. oryzae pathotype Triticum could also be associated with other Poaceae plant species invasive to wheat fields (Figure 1).
Branch II, with 88.1 bootstrap support, grouped the isolates of P. zingibericola obtained from invasive plants [12.0.149 (from Urochloa spp.), 12.0.212(from Panicum maximum) and 12.0.595i(from Chloris distichophylla)] and the isolate P. zingibericola, whose sequences were obtained from the GenBank.Only Zingiber officinale had been reported as a host of P. zingibericola, in the Réunion Island (Klaubauf et al. 2014), and, so far, there have been no reports of this pathogen infecting invasive plant species in Brazil.Branch III, with 100.0 bootstrap support, grouped the isolates of P. pennisetigena (13.0.024i; 13.0.012i;13.0.001i;12.0.402;12.0.358;12.0.324;12.0.002i;12.0.100)and the isolate whose DNA sequences were obtained from the GenBank.The P. pennisetigena group was found in the invasive plant species Urochloa spp., Cenchrus echinatus and Panicum maximum.The Poaceae C. echinatus had already been described by Klaubauf et al. (2014) as a host of P. pennisetigena, as well as C. ciliaris, Echinochloa colona and pearl millet (Pennisetum glaucum).
Significant differences were detected among Pyricularia species according to the blast severity observed in signal grass cv.Piatã (Table 2; Figure 2).
Pyricularia zingibericola was the most aggressive species to signal grass (Figure 3A), causing, on average, 24.1 % of infected leaf area.Among the other species, in decreasing order of aggressiveness, P. oryzae from invasive plants and wheat caused an average of 14.8 % disease severity, differing from P. grisea (8.8 %) and P. pennisetigena (5.2 % of infected leaf area).
One of the factors that could explain the higher aggressiveness of P. zingibericola to signal grass is the origin of a few isolates that came from this same host, thereby explaining its better adaptability to this plant species.
All four species of Pyricularia tested were pathogenic to barley cv.BRS Korbel (Figures 2B  and 3B; Table 2), but P. zingibericola was the most aggressive one (mean of 18.7 % of diseased leaf area).There was no significant difference among the groups of isolates from P. pennisetigena, P. grisea and P. oryzae, regarding the disease severity on young barley plants, with an average leaf blast severity corresponding to 12.6 %, 12.0 % and 11.4 %, respectively.Indeed, innumerable barley cultivars have been considered susceptible to pathogens of the Pyricularia genus, especially P. oryzae (Marangoni et . 2013).However, there is variation in the level of blast resistance among genotypes, with some having high levels of resistance to P. oryzae (Marangoni et al. 2013).
For wheat cv.Anahuac 75, P. pennisetigena was the most aggressive species to young plants, with an average of 26.6 % of diseased leaf area.On the other hand, the group of P. oryzae and P. zingibericola caused similar damage, with an average disease severity of 20.0 % and 18.3 % of infected leaf area, respectively.Pyricularia grisea was not pathogenic to wheat cv.Anahuac 75 (Figures 2 and 3C; Table 2).
Possibly, this is the first report suggesting that P. pennisetigena and P. zingibericola may cause leaf blast on signal grass, barley and wheat.However, from an evolutionary point of view, it is unknown how recent the adaptation of the two Pyricularia species to these distinct hosts is.Yet, from an ecological point of view, it is still unknown the relative importance of P. pennisetigena and P. zingibericola, in relation to the other species associated with blast disease already described for signal grass, barley and wheat, in the Brazilian agroecosystem.
Finally, this study has also shown that the P. oryzae pathotype Triticum has a host range wider than wheat, barley, rye and oat (Igarashi et al. 1986, Urashima et al. 1993, Anjos et al. 1996, Goulart et al. 2003).
In particular, considering the host range of the P. oryzae pathotype Triticum, it is possible to highlight the importance of invasive plants to wheat fields as an initial inoculum source of wheat pathogens, thereby confirming the observations by Urashima et al. (1993).Although ear infection is the most destructive form of wheat blast disease (Goulart & Paiva 2000, Torres et al. 2009), little is known about the origins of the initial inoculum source.It is plausible that the initial inoculum of the pathogen produced in the leaves of invasive grasses has an important epidemiological role in the incidence of wheat blast on ears.
Among the invasive plant species, whether native or not, Urochloa spp., Panicum spp.and Cynodon spp.stood out as important additional hosts, due to their wide geographical distribution as cultivated forages in Brazil (Zimmer et al. 2012).Urochloa spp., the most cultivated forage in Brazil, was the host of P. grisea, P. pennisetigena and P. zingibericola, and presented high susceptibility to the P. oryzae pathotype Triticum.Therefore, extensive grass pasture fields cultivated with plants of the Urochloa genus, especially in the Brazilian Savannah, would represent a potential permanent source of initial inoculum between the wheat growing seasons, thereby keeping the inoculum of the P. oryzae pathotype Triticum active.***, * and ns Significant at p ≤ 0.001, p ≤ 0.05 and non-significant, respectively, by the F test.

Figure 1 .
Figure 1.Reconstruction of the phylogenetic relationship among four species of the Pyricularia genus, based on sequences of the actin (ACT) and calmodulin (CAL) genes concatenated.Maximum likelihood (ML) implemented in the PhyML algorithm was used.The internode support for the branches was tested by 10,000 bootstrap resamplings of the data.

Table 1 .
(Maciel et al. 2013respective Pyricularia species obtained from grass plants invasive to wheat fields in 2012 and 2013, as well as their accession number for the actin (ACT) and calmodulin (CAL) gene sequences deposited at the NCBI GenBank.Pyricularia pennisetigena and P. zingibericola from invasive grasses infect signal grass, barley and wheat transferred to ten other Petri dishes with oatmeal agar medium, aiming at the production of fungal inoculum.The fungal colonies were kept for 15 days under the same incubation conditions(Maciel et al. 2013).

Table 2 .
Variance analysis table for testing the effects of the experiment, species and pathogen isolates on the aggressiveness of Pyricularia spp. to signal grass, barley and wheat.Pyricularia pennisetigena and P. zingibericola from invasive grasses infect signal grass, barley and wheat Pyricularia zingibericola and P. pennisetigena are pathogenic to signal grass, barley and wheat, with varying levels of aggressiveness between the species.Pyricularia zingibericola is the most aggressive species to signal grass and barley.2. For young wheat plants, P. pennisetigena is the most aggressive species, followed by the P. oryzae pathotype Triticum.On the other hand, P. grisea from D. sanguinalis or from Urochloa spp.do not infect wheat.3. The phylogenetic analysis of the concatenated ACT and CAL regions sustains the genetic differences among Pyricularia zingibericola, P. pennisetigena, the P. oryzae pathotype Triticum and P. grisea.4. Urochloa spp.represents a permanent source of initial inoculum of the P. oryzae pathotype Triticum between wheat growing seasons.