Barberry plays an active role as an alternate host of Puccinia graminis in Spain

Abstract Stem rust, caused by Puccinia graminis, is a destructive group of diseases. The pathogen uses Berberis species as alternate hosts to complete its life cycle. B. vulgaris and the endemic species B. hispanica and B. garciae are present in Spain. The objective of this study was to investigate the functionality of the indigenous barberry as alternate hosts. Field surveys were conducted in 2018 and 2019 in Huesca, Teruel and Albacete provinces of Spain. Aecial samples on barberry were analysed via infection assays and DNA analysis. B. garciae was predominant in Huesca and Teruel provinces, often found in the field margins of cereal crops. Aecial infections on B. garciae were observed in May and uredinial infections on cereal crops in June. Scattered B. hispanica bushes were occasionally found near cereal crops in Albacete, where aecial infections on B. hispanica were observed in June when most cereal crops were mature. Infection assays using aeciospores resulted in stem rust infections on susceptible genotypes of wheat, barley, rye and oat, indicating the presence of the sexual cycle for P. graminis f. sp. tritici, f. sp. secalis and f. sp. avenae. Sequence analyses from aecial samples supported this finding as well as the presence of Puccinia brachypodii. This study provides the first evidence that indigenous Berberis species play an active role in the sexual cycle of P. graminis under natural conditions in Spain.


| INTRODUC TI ON
Stem rust of cereal crops and grasses, caused by Puccinia graminis, is a destructive group of diseases that has caused human misery for more than a millennium (Kislev, 1982). P. graminis comprises specialized forms (or formae speciales, ff. sp.) that attack specific cereal crop and grass species, such as the form P. graminis f. sp. tritici (Pgt) that mainly attacks wheat. Barberry (Berberis spp.) serves as the aecial host where the sexual cycle of the fungus is completed. Many Berberis spp. are known to be susceptible to P. graminis (Levine & Cotter, 1932). Breeding stem rust-resistant wheat varieties and large-scale removal of common barberry (Berberis vulgaris) from major wheatgrowing regions resulted in effective control of the wheat stem rust in North America and western Europe during the first two thirds of the twentieth century (Peterson et al., 2005). However, over the last two decades some countries have experienced a resurgence of stem rust after many decades of quiescence (Berlin et al., 2015;Olivera Firpo et al., 2017;Olivera et al., 2015Olivera et al., , 2019Saunders et al., 2019).
Part of this resurgence was caused by the evolution and continuing spread of Sr31-virulent races, commonly known as the Ug99 race group, which began in East Africa in 1998 and radiated to adjacent countries and beyond over ensuing years. In 2013, multiple Pgt races with novel virulence combinations were detected from samples collected in central Germany where wheat stem rust outbreaks occurred (Olivera Firpo et al., 2017). In 2016, a highly virulent race, TTRTF, detected first in a sexual population from Georgia (Olivera et al., 2019), caused a severe outbreak in durum wheat in Sicily, Italy (Bhattacharya, 2017). Stem rust infections on cereals were observed in 10 European countries in recent years (Hovmøller et al., 2020), including the United Kingdom (Lewis et al., 2018), Denmark and Sweden (authors' unpublished data).
New genetic variants of P. graminis, largely exampled by f. sp. tritici, can arise by both asexual and sexual mechanisms. Asexual variation independent of the presence of alternate hosts is caused by mutation of single avirulence factors or somatic recombination that is not well understood and more complex than simple nuclear exchange (Park & Wellings, 2012). The role of sexual recombination in generating genetic variability in P. graminis has been well documented (Craigie, 1927;Roelfs, 1982;Stakman et al., 1930).
According to Roelfs (1982) and Peterson et al. (2005), the massive eradication of barberry in the United States from 1918 to 1974 had a large impact on reducing genetic variability and increasing the stability of Pgt races found in the US Great Plains. This topic has regained attention recently. The resurgence of stem rust in Europe after decades of near absence turned attention to barberry, with localized epidemics and identification of multiple pathogen races associated with the presence of barberry (Berlin et al., 2013;Olivera Firpo et al., 2017;Olivera et al., 2019;Saunders et al., 2019). Multiple races of Pgt with highly diverse virulence combinations were recovered from the Caucasus region (Olivera et al., 2019) and central Asia (Berlin et al., 2015). This implied that sexual recombination of Pgt on barberry is also common in these regions. Sexual recombination has two genetic aspects apart from the role of telia in cross-season survival of the pathogen. First, recessive alleles for virulence carried by heterozygous avirulent individuals can become homozygous virulent; and secondly it generates new gene combinations (i.e., races), some of which might overcome deployed resistance gene combinations and therefore will be of a selective advantage.
Most of our knowledge on the role of barberry in wheat stem rust pathogen variation and disease epidemiology relates to common barberry, B. vulgaris. However, there is a wide range of Berberis spp.
(and interspecific hybrids) and knowledge on their reaction to Pgt and association with cereal crops is limited. Although evidence is lacking, it is reasonably expected that there is genetic variation within barberry species and populations in regard to infection by P. graminis.
Finally, Berberis spp. are alternate hosts for different graminaceous rust pathogens and the presence of pycnia or aecia on barberry plants is not sufficient to conclude a connection between cereal stem rust and barberry. Because the morphologies at the aecial stage of different Puccinia spp. infecting barberry are very similar, infection assays via inoculation experiments or DNA-based diagnosis are needed to identify specific rust species in an aecial sample.
Spain is one of the major wheat-producing countries in western Europe. Wheat occupied nearly 2 million hectares in 2018, 82% bread wheat and 18% durum wheat, with total production of 8 million tonnes (MAGRAMA, 2020). Wheat stem rust has not been considered a major problem in Spain in recent decades. A significant factor was the widespread adoption of early maturing stem rustresistant varieties in the 1960s, after which reports of stem rust be-  (Monserrat, 1970). samples collected from wheat during the same years. Salazar and Brañas (1973) also reported that races recovered from rusted wheat samples collected near barberry bushes were different from races identified from samples collected in fields without adjacent barberry.
The unusual virulence combination (including virulence for Sr31) of a Pgt isolate derived from a wheat stem sample collected near barberry in Monteagudo del Castillo (Teruel province, Spain) further implicated the sexual cycle in barberry . The objective of the present study was to investigate the functionality of indigenous Berberis spp. in Spain as the alternate hosts for P. graminis.  Table S1 shows the sampling calen- Huesca and Teruel provinces are located in the Ebro River basin area in north-east Spain (Figure 1). The Ebro River basin has a typical Mediterranean continental climate with an annual average temperature of 9 to 11°C, cold winters below 0°C and hot summers exceeding 30°C during June to September. Annual average rainfall in this region ranges from 300 mm in the central basin (Lleida) to more than 1000 mm in the mountainous extremes of the Pyrenees in the north (Huesca) and Iberian System in the south (Teruel). Winter and summer are the driest periods and over 70% of the rainfall occurs in spring and autumn. Albacete province is in south-eastern Spain at an altitude of 1000 m a.s.l. and has a mild Mediterranean continental climate with average temperature of 14.2°C with short cold winters and summer days exceeding 35°C (Table   S2). Annual average rainfall in this area is much lower than that of the northern central Pyrenees and Iberian System range.

| Field surveys
During surveys, the GPS coordinates at each location were recorded with a Garmin GPSmap 60CSx instrument. The survey data during the survey were later keyed to species based on morphological parameters, as described in Flora Iberica (Lopez González, 1986).
Sampled grasses were initially keyed to respective species by the authors following classifications by Romero (2011). Identifications

| Inoculation assay on identification series
A set of genotypes including wheat (Triticum aestivum 'Morocco' and Line E), barley (Hordeum vulgaris 'Hiproly'), rye (Secale cereale 'Prolific') and oat (Avena sativa 'Marvelous') was used for initial inoculation experiments. These cultivars are known to be "universal" susceptible genotypes of the cereal crop species that serve as the aeciospore infection hosts to provide a preliminary identification of Pgt, f. sp. secalis (Pgs, rye stem rust pathogen) and f. sp. avenae (Pga, oat stem rust pathogen). Bulked barberry leaf samples bearing aecia were used to inoculate seedling plants of the above genotypes following the procedure of Jin et al. (2010).
This inoculation method allows the release of aeciospores from aecia placed above the seedlings in a moisture-saturated environment in a dew chamber. When aecial samples were small, aeciospores were collected into gelatin capsules, suspended in mineral oil and sprayed onto the seedling plants following a procedure that was similar to urediniospore inoculation (Jin et al., 2007). An infec-

| DNA extraction and ITS sequencing of aecial samples
Genomic DNA was extracted from single aecial pustules using the OmniPrep DNA extraction kit (G-Biosciences), following the manufacture's protocol for fungal tissues. A segment of approximately 1300 bp containing the 5′ end of the 18S rRNA, complete internal transcribed spacer (ITS) region and 5′ end of the 28S rDNA was amplified using the primer pair ITS1-F (Gardes & Bruns, 1993) and RUST1 (Kropp et al., 1995). PCRs were conducted in total reaction volumes of Raw sequences were assembled using the de novo assemble function in Geneious Prime (http://www.genei ous.com). After trimming the partial 18S and 28S rRNA regions, sequences from the eight recombinant plasmids were multi-aligned and consensus sequences for each sample were generated. Final error-corrected consensus sequences were deposited in GenBank (accession numbers in Table S3).

| RE SULTS
3.1 | Barberry species, their distribution and cereal production system B. garciae was the predominant species in Huesca and Teruel provinces ( Figure 1). Some rare hybrids of B. garciae and B. vulgaris were also identified at Hostal de Ipiés in the Jaca area (Table 1).
In both provinces, B. garciae was found at altitudes from 600 to 1500 m a.s.l. where cereal crops are widely grown. Barberry bushes were often found at the field margins and in very close proximity (0-25 m) to cereal crops. Various grasses were common in the field margins together with barberry bushes (Figure 2). In the Jaca area of Huesca province (Figure 2a), barberry was present as isolated plants or in clusters at the field margins of fewer than 15% of fields. In Teruel province, especially the Gúdar area, barberry was very common and found in the margins of more than 50% of the fields, often forming large clusters. In this area, barberry was frequently in close proximity to cereal fields (Figure 2b).
At the Campo de Montiel site in Albacete province in the south (Figure 2c), B. hispanica was found at higher altitudes, approximately 1000 m a.s.l. Although barberry bushes at this site were scattered and found in less than 5% of the area, they were close to cereals crops (Figure 2c). B. hispanica can be found up to 2500 m a.s.l. (López González, 1986), but cereal crop cultivation rarely occurs at that altitude.
The most widely grown cereal crops at the surveyed sites were wheat, barley and triticale, with occasional oats and rye. The proportions of each crop differed between sites ( Table 1). Barley and wheat were predominant followed by triticale in the Jaca area in Huesca province ( Table 1). Triticale was the most widely grown crop in the Albarracín area in Teruel province, followed by barley and wheat (  Grass species that could potentially serve as accessory hosts for cereal stem rust pathogens and those with stem rust infections are given in Table 1 Table 1 because they were not common.

| Identification of Puccinia spp. based on ITS
Of the total 51 aecial samples received at CDL in 2018 and 2019, the 1.3 kb region containing partial 18S, complete ITS sequence and partial 28S rDNA was successfully amplified and sequenced for 22 samples. BLAST analysis showed 21 of 22 samples had best hits with P. graminis and shared very high similarity (E-value: 0%, ID: 99.1%-100%; Table S3). One sample, 18SPA066, shared high simi-

| DISCUSS ION
There has been growing global concern regarding the re-emergence of wheat stem rust after many decades of quiescence. Once feared as the "bubonic plague of wheat", this disease was deemed to be under control until race Ug99, with virulence to the widely deployed resistance gene Sr31, caused serious outbreaks in East Africa and made an estimated 80% of the world's wheat germplasm vulnerable to stem rust (Singh et al., 2011). In the last 20 years, multiple stem rust outbreaks and epidemics have been reported in Asia, Africa and in Europe, which had not experienced significant stem rust outbreaks in the previous 50 years (Olivera Firpo et al., 2017;Olivera et al., 2015Olivera et al., , 2019Saunders et al., 2019). As several countries in Europe and other parts of the world were experiencing a resurgence of stem rust in wheat, more intensive searches revealed a concurrent increase in the prevalence of common barberry (B. vulgaris) in wheatgrowing regions and provoked questions regarding the potential role TA B L E 2 Production of uredinia on cereal crop species and genotypes inoculated with aeciospores from infected barberry  (Ahrendt, 1961). Although the ITS sequence analysis cannot distinguish between P. Identification of the host virulence aspects of uredinial samples from grasses will be a necessary adjunct to field surveys of infections on both cereals and potential alternate hosts. The fact that fallow and non-cereal crop rotations observed in Teruel did not prevent stem rust infection in cereals in this region reinforces the idea that grasses may have played an important role in sustaining infections without the presence of cereal crops. If this role could be confirmed, management of weedy grasses near barberry plants along the field margins would provide a potential novel approach for stem rust control.
It is well established that the sexual cycle of the stem rust pathogen on barberry allows the development of new races following genetic recombination among loci (Jin, 2011;Roelfs, 1982). However, there must be a coincidence of aeciospore release and the presence of a receptive telial host. Earlier maturing autumn-sown cereals in some regions were a major contributor in reducing the risk of stem rust. The ubiquitous co-occurrence of susceptible barberry adjacent to cereal crops and accessory grass hosts observed in this study is concerning. Although pathogen strains with novel virulence or unique virulence combinations generated through sexual cycles may not be favoured by selection in the short term, avirulence/virulence polymorphisms will continuously evolve and be sustained in the local population. This may incite epidemics many years later, such as the epidemics of race 15B in the United States (Jin, 2011).
The widespread occurrence of barberry adjacent to cereal crops is equally concerning for stripe (yellow) rust pathogens of wheat, triticale and barley. Although P. striiformis is not currently known to undergo sexual reproduction in Europe (Rodríguez-Algaba et al., 2021) and P. striiformis was not detected from aecial samples in this study, there has been increasing concern that the sexual stage of P. striiformis is already established in Europe (Lewis et al., 2018).

DATA AVA I L A B I L I T Y S TAT E M E N T
The ITS sequence data of aecial samples report in this this article are deposited in GenBank under the accession numbers listed in