RETRACTED ARTICLE: Alternate hosts and their impact on genetic diversity of Puccinia striiformis f. sp. tritici and disease epidemics

We, the Editor and Publisher of Journal of Plant Interactions have retracted the following article: Journal of Plant Interactions: Sajid Mehmood, Marina Sajid, Lili Huang & Zhensheng Kang (2020) Alternate hosts and their impact on genetic diversity of Puccinia striiformis f. sp. tritici and disease epidemics, Journal of Plant Interactions, 15:1, 153-165, DOI: 10.1080/17429145.2020.1771445 . The above article has been retracted as a result of the Editor and the Publisher determining, through post-publication review, that there is significant overlap with a previously published article by the same authors, Mehmood S, Sajid M, Zhao J, Huang L, Kang Z. Alternate Hosts of Puccinia striiformis f. sp. tritici and Their Role. Pathogens. 2020; 9(6):434. . We have had the full cooperation of the authors during our investigations. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as “Retracted”.


Introduction
Wheat is the most cultivated cereal crop, susceptible to many diseases. One of the most serious threats to the wheat crops is fungal diseases comprising of rusts causing severe yield losses worldwide (Wellings 2011). Stem rust or black rust, caused by Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn (Pgt), leaf rust or brown rust, caused by Puccinia triticina Erikss. (Pt) and the wheat stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici (Pst), are historically crucial economic wheat diseases that occur in almost all wheat-growing areas in the world (McIntosh et al. 1995;Singh et al. 2004). The early history of mankind is full of fears and threats of these devastating rust pathogens and their aggressiveness to destroy wheat crops. Since the discovery of rust pathogens, numerous investigations have been conducted on their life cycles for the management of these diseases. The tenacity of rust fungi as a significant wheat disease throughout the wheat-growing areas in the world is attributed to the specificity of the pathogen, for example, production of a large number of spores and their inter and intracontinental wind dissemination, their ability to change genetically, resulting in new races with increased virulence diversity (Brown and Hovmøller 2002). Generally, the disease occurs at high elevations in the northern and southern areas of temperate regions. Recently, the wheat stripe rust disease epidemics are reported in warmer areas, where this disease was considered as unimportant or absent (Hovmøller et al. 2010), causing food safety issues worldwide (Luo et al. 2015). Hot summers and dry seasons are most threatening to the wheat stripe rust pathogen survival. Preventive fungicide sprays can control the pathogen. Resistant wheat cultivars are the most effective, economical, and environmentally friendly approach to combat with the wheat stripe rust pathogen. However, the Pst population is highly dynamic and variable which makes it difficult to develop highly resistant wheat cultivars with durable resistance (Luo et al. 2015). In the US Pacific Northwest, barberry is essential for the wheat stem rust pathogen but does not play a role for the wheat stripe rust pathogen (Cheng and Chen 2014;Wang and Chen 2015b). Barberry may serve as an alternate host for the wheat stripe rust pathogen in Pakistan and the Himalayan region under natural conditions (Ali et al. 2010;2014a;Mehmood et al. 2019). In western Asia and eastern Africa, barberry plants have been found, but their association with the wheat stripe rust disease epidemics has not been confirmed (Jin et al. 2010;Jin 2011). So far, the only evidence of natural infection of barberry by Pst has been obtained in China, but at a low frequency Zhao et al. 2013). Similarly, Pst has not been found on Berberis spp. in southeastern Sweden, but Pgt is common on the alternate host plants in the region (Berlin et al. 2012).
Genetic techniques, in the past ten years, have witnessed sizeable advances in understanding the plant-microbe interaction. The wheat stripe rust disease is one of the most substantial threats to wheat production worldwide (Beddow et al. 2015). It is an obligate, biotrophic parasite, having a macrocyclic heteroecious sexual life cycle. A macrocyclic life cycle comprises of uredinial, telial, basidial, pycnial and aecial stages. Pst is highly specific to their host plants (Zhao et al. 2016a). More than 3, 000 species of Pucciniaceae, with a high diversity of host specification and a different number of spore stages during the life cycle, have been found in Puccinia (Van der Merwe et al. 2008;Liu and Hambleton 2010). Puccinia spp. is one of the 'top ten' fungi in molecular plant pathology, and cereals (wheat, triticale, rye, and barley) and some grasses (Brown and Hovmøller 2002;Dean et al. 2012).
The genetic diversity of Pst in Europe, Australia, and North America has indicated a clonal population structure of the pathogen without sexual recombination (Hovmøller et al. 2002). In contrast, the Pst populations of Gansu Province, China, have been found to have high genetic diversities and produce abundant telia, indicating possible sexual recombination in this area (Mboup et al. 2009;Duan et al. 2010). Jin et al. (2010) reported barberry (Berberis spp.) as an alternate host for Puccinia pseudostriiformis (Syn. Puccinia striiformis f. sp. poae) (Liu and Hambleton 2010); under natural conditions in Minnesota, the United States and Pst under controlled conditions. The possible role of barberry as a sexual host of Pst has attained much importance, particularly in the United States, China, and Pakistan (Zhao et al. 2013;Ali et al. 2014a;Wang and Chen 2015b;Zhao et al. 2016a). Mahonia aquifolium, under experimental conditions, has also been identified to be susceptible to Pst. Based on different effects by the microorganisms on plants, their relationship can be pathogenic, saprophytic, or beneficial. However, in all types of interaction with plants the pathogens use the same strategies and interaction mechanism of gene-for-gene model, where each gene for resistance in the host plant has a corresponding gene for avirulence in the pathogen, and each gene for virulence in the pathogen there is a corresponding susceptible gene in the host plant. The wheat stripe rust-pathosystem follows the gene-forgene concept (Flor 1971). An incompatible interaction (or resistance) is observed when a resistant isolate carrying an avirulence gene encounters a plant possessing a corresponding resistance gene. A compatible or susceptible reaction is observed when an avirulence gene or a resistance gene does not function or is absent in the host plant. Nature of the host resistance and capability of the pathogen are important factors to understand the genetics of avirulence/virulence. Based on this concept, it is assumed that avirulence is dominant for virulence for Pst (Rodriguez-Algaba et al. 2014). The most economical and preferred strategy to control Pst is the use of genetically resistant R genes. Seedling resistance genes confer resistance against all wheat rusts encode classic nucleotide-binding site-leucine-rich repeat (NBS-LRR) R proteins that recognize effector proteins, present in the cytoplasm and they stop pathogen multiplication by triggering a defense response (Ellis et al. 2014;Steuernagel et al. 2016). Hundreds of disease resistance (R)-gene loci have been genetically mapped to wheat chromosomes, however only a small number of yellow rust resistance genes (Yr) have been isolated so far (Yr10, Yr18, Yr36, Yr46, Yr5/YrSP, Yr7, and Yr15) (Klymiuk et al. 2018;Marchal et al. 2018). Yr10 is the best example of a cloned classic R gene against Pst, which confers resistance to many Pst isolates worldwide. However, several Pst isolates virulent to Yr10 have been identified (Liu et al. 2014). The successful cloning of Yr15, another broad-spectrum R-gene discovered in the 1980s, derived from wild emmer wheat, which encodes a putative kinasepseudokinase protein, designated as wheat tandem kinase 1, comprising a unique R-gene structure in wheat, was reported by Klymiuk et al. (2018). Although few Pst races were reported to be virulent on Yr15 in the 2000s (Hovmøller and Justesen 2007). With time, the evolution of new virulent isolates of Pst is a characteristic of classical resistance genes, and it is known as the 'boom and bust' cycle. Adult plant resistance genes delay pathogenic infection and spore production. These nonclassical R genes cause resistance allelespecific protein variants that are molecularly unrelated to NBS-LRR proteins (Ellis et al. 2014). Yr18, Yr46, and Yr36 are good examples of these genes (Ellis et al. 2014;Gou et al. 2015;Moore et al. 2015). The strategy to combat Pst using a combination of classic and nonclassic R genes has been found very useful because it slowdowns the life cycle of the pathogen and reduces its population size. However, the durability of these genes may be affected by the global genetic diversity of the pathogen as a result of sexual or asexual recombination (Schwessinger 2016). The breeders and pathologists strongly believe in the genetic control of this disease using yellow rust resistance genes (Yr), and they are working on the identification of such resistant genes over the last 100 years (McIntosh et al. 2013). In recent years, the use of DNA-based technology has made it possible to clone first Yr resistant genes, identification of its complete life cycle, identification of its center of diversity in the Himalayan region of Pakistan, past global migration routes and patterns, and most importantly the provision of the drafting of Pst genomes (Schwessinger 2016). In this review, we have focused on i) the research progress on the identification of alternate hosts to Pst in the world; and ii) their possible role in the pathogenic diversity, and iii) recent research progress in understanding the genetics of Pst.

The wheat stripe rust disease
Wheat rusts have created major famines throughout human history, causing substantial economic losses (Roelfs et al. 1992). Currently, the most severe rust disease is the wheat stripe rust disease, which can cause more than 60% yield losses under favorable conditions (Villaseñor-Espín et al. 2009;Wellings 2011;Ellis et al. 2014). Further, the non-random distribution of disease pattern, the stripe rust uredinia differs from other wheat rusts for being smaller and for producing clear yellow urediniospores. Urediniospores are asexual, dikaryotic spores produced in a uredinium of a rust fungus on a primary host. In case of severe epidemics, stripe rust uredinia can affect all green parts of the plant, including leaves, spikes, spikelets, glumes, awns, and damaging the wheat seeds. With the increase in temperature and maturity of the plants, the further production of urediniospores comes to an end. The uredinia start converting into teliospores (Chen 2005). Teliospores are thick-walled resting spores, in which karyogamy occurs; it germinates to form a promycelium from which haploid basidiospores are produced. Previously it was thought that the wheat stripe rust disease occurs only in the temperate regions and areas of high lands due to the requirement of lower temperatures for the development of the disease (Chen 2005). However, the wheat stripe rust disease epidemics have also been reported in areas of high temperatures, which were formerly considered too warm for the wheat stripe rust disease epidemics (Milus et al. 2009;Hovmøller et al. 2010). The scientific classification of the stripe rust pathogen is given in Table 1. A general classification of B. vulgaris is provided in Table 2.
Pst can undergo long-distance dispersal and it has caused numerous invasions worldwide (Zadoks 1961;Wellings and McIntosh 1990;Markell and Milus 2008;Hovmøller et al. 2015). Several cases of economically important incursions have been reported for Pst but only recently the origin of these was confirmed (Ali et al. 2014a;Hovmøller et al. 2015). In the early twentieth century, Pst was reported for the first time in North and South America (Carleton 1915;Rudorf and Job 1934), most likely spreading from NW Europe (Hovmøller et al. 2011;Ali et al. 2014a) and it was introduced accidentally in Australia from NW Europe in 1979 (Wellings and McIntosh 1990) through human transmission (Wellings 2007). Pst strains detected in South Africa in 1996 were later shown to be genetically related to populations in the Middle Eastern and Mediterranean regions, which were possibly spread by wind (Boshoff et al. 2002;Ali et al. 2014a). To one side from these recent incursions in previously uncolonized areas, Pst has also been important in the context of invasion and recolonization through the emergence of new races. For example, since 2000, the emergence of two high temperature-adapted aggressive strains, PSTS1 and PSTS2, in the geographical expansion of Pst epidemics into Western Australia and the South Eastern US, where the disease was not previously considered a problem (Chen 2005;Milus et al. 2009). Since 2011, invasive strains of the 'Warrior' and 'Kranich' have largely replaced the pre-existing northwest European Pst populations (Hovmøller et al. 2015;Hubbard et al. 2015).
In recent years Pst has been reported in the areas of US (especially Pacific North West), East Asia (China-northwest and southwest), Oceania (Australia, New Zealand), South Asia (India, Pakistan, and Nepal), the Arabian Peninsula (Yemen) Western Europe (east England), and East Africa (Ethiopia, Kenya) (Waqar et al. 2018). In the last two decades, the emergence of more aggressive races of Pst having the ability to cause high epidemic potential even in warmer regions (Khanfri et al. 2018) is generally the result of mutation, somatic hybridization, and sexual recombination. The potential role of alternate hosts in pathogenic diversity is of much importance. However, it is still unknown by which mechanisms new races evolve. The high reproduction ability, long distances dissemination, adaptation to different environmental conditions, and several host species make Pst a highly diversified pathogen (Wan et al. 2017). The threat of new virulent races of Pst pathogen emphasizes the need to understand the mechanisms involved in the genetic diversity of Pst and the role of aecial hosts in sexual reproduction to encounter the possible attacks of Pst in the future (Khanfri et al. 2018). Historical perspectives of the widespread wheat stripe rust disease epidemics have been discussed in detail by Chen (2020).

Pst life cycle
The life cycle of Pst consists of five different spore stages on two phylogenetically distinct host plants, a cereal as the primary host or asexual host and Berberis spp. as the alternate host or sexual host (Jin et al. 2010). The dikaryotic (N + Nʹ) single-celled urediniospores appear on the primary host through the breakage of epidermal cells, and each uredinia harboring yellow-colored numerous urediniospores. The repeated asexual cycles on the primary host may cause wide-scale epidemics on the cereal hosts ). The individual uredinia, clustered in lesions, form typical stripes on the leaves of adult plants produce spores successively 10-18 days after infection. Each lesion expands longitudinally by the formation of new uredinia. With the start of senescence of infected leaves, P. striiformis starts producing telia resulting in the creation of many two-celled oblong-clavate teliospores. These teliospore cells contain a diploid nucleus (N + Nʹ) formed by karyogamy. The germinating teliospores produce ellipsoid haploid (N) basidiospores. Basidiospores are uninucleate or binucleate haploid spores produced from a promycelium resulted from a germinating diploid teliospore. These basidiospores cause infection on the alternate host (e.g. barberry), resulting in oblong-shaped pycniospores (N) on the upper side of the leaf, followed by the formation of dikaryotic (N + Nʹ) aecia on the lower side of the leaf. Pycniospores are haploid (N), sexually derived spore (spermatium) formed in a pycnium (spermogonium) of a rust fungus. Finally, the aeciospores infect primary host resulting urediniospores onto wheat leaves, which lead to typical yellow rust urediniospores. The life cycle may take place in two adjacent growing seasons of the primary host. P. striiformis is an obligate biotroph, depending on a living host for growth and reproduction (Hovmøller et al. 2011;Rodriguez-Algaba et al. 2014).
The sexual phase starts when two-celled teliospore germinates, producing basidia containing basidiospores, attached to a sterigmatum. These haploid basidiospores (2*N) make infection on barberry leaves, resulting in the formation of pycnia (N), covered with pycnial nectar, formed in the adaxial side of the barberry leaf. The haploid pycniospores of (-) and haploid hyphae (+) fuse together through plasmogamy and form aeciospores (2N) on the abaxial side of the barberry leaf (Rodriguez-algaba et al. 2017). Aeciospores are dikaryotic spores produced in the cup-shaped aecium of a rust fungus on an alternate host. The asexual infection process in cereal hosts starts via a urediniospore germ tube, penetrating the stoma and then differentiating into a substomatal vesicle. This vesicle forms typically 2-3 primary infection hyphae, which develop haustorial mother cells. These cells are separated by a septum from the respective hyphae. These haustorial mother cells penetrate the plant cell walls and form haustoria, which are highly specialized structures that represent the primary interface between host and pathogen. These haustoria play an essential role in water and nutrient acquisition from the host tissues and in signaling between host and pathogen by secretion of effector molecules, such as avirulence gene products. Young haustoria had a spherical shape, whereas mature haustoria appeared more branched. These branched haustoria allow the fungus to extend the area of the contact zone with the host and may uptake nutrients more efficiently (Hovmøller et al. 2011). Basidiospores cannot infect (primary hosts) but can infect alternate hosts (barberry) to complete the sexual cycle. Basidiospores infect epidermal cells through direct penetration while urediniospores infect through host stromata. The host range of the Pst with different spore stages is given in Table 3. The complete life cycle of Pst is shown in Figure 1.

Alternate hosts of Pst
Alternate hosts are different from primary hosts. The complete life cycle of Pst was demonstrated by Jin et al. (2010) declaring barberry as the alternate hosts for the wheat stripe rust pathogen. Wang and Chen (2015a) identified M. aquifolium as an alternate host for Pst. Similarly, Zhao et al. (2013;2016a) identified several barberry species as alternate hosts of Pst in China under controlled conditions. Mehmood et al. (2019) inoculated seven barberry species collected from the Himalayan region of Pakistan and found them susceptible hosts to Pst under greenhouse conditions. The general procedure adopted for growing barberry plants in the greenhouse and artificial inoculation of these barberry plants using germinating teliospores is described in schematic Figure 2. A comparison of controlled conditions used for artificial inoculation of alternate hosts of Pst is summarized in Table 4. The discovery of alternate hosts for Pst was important for a better understanding of the pathogen lifecycle and the mechanisms of genetic diversity. Berberis spp. had been well known as alternate hosts for P. graminis f. sp. tritici for more than a century (Roelfs 1982). Before the discovery of Berberis spp. as a sexual host of the wheat stripe rust pathogen, it was challenging to study virulence variation of Pst. Different Puccinia species can have similar cereal and grass hosts but generally have different alternate hosts. Grasses and cereals harbor a massive of the stripe rust fungi, containing more than 380 species in genera of Puccinia and Uromyces, and only some of them are heteroecious (Cummins 1971). Using combined methods of spore morphology and sequences of the internal transcribed spacer (ITS) and beta-tubulin DNA regions, the stripe rust fungi infecting bluegrass and orchard grass are renamed as P. pseudostriiformis M. Abbasi, Hedjaroude, and M. Scholle, and P. striiformoides M. Abbasi, Hedjaroude, and M. Scholle, respectively (Abbasi et al. 2004;Liu and Hambleton 2010). P. striiformis on grass species in genera of Aegilops,  Stage III (the Telial stage) appear on its primary host (e.g. wheat). The urediniospore (N + Nʹ) appear on green parts of the plant (e.g. leaves) and at senescence, these urediniospores may convert into two-celled teliospores (N + Nʹ). Stage IV (the Basidial stage) the two-celled teliospores germinate with a basidium and produce four basidiospores (2*N).

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Elymus, Hordeum, and Triticum, should be recognized as P. striiformis sensu stricto (Liu and Hambleton 2010) until the discovery of alternate hosts for Pst and P. pseudostriiformis (syn. P. striiformis f. sp. poae) on Poa pratensis (Jin et al. 2010). All the early attempts to find aecial hosts for Pst was unsuccessful (Tranzschel 1934;Straib 1935;Eriksson and Henning 1894;Stubbs 1985), however Mains (1932) made the correct speculation, proven more than 70 years later, that Berberis and Mahonia could be alternate hosts of Pst for its relatedness to P. koeleriae, P. arrhenatheri, and P. montanensis. Jin et al. (2010) identified a stripe rust fungus by inoculating wheat, barley, oat, rye, and Kentucky bluegrass with aecium-bearing leaf samples collected from naturally infected plants of Berberis chinensis and Berberis koreana in an arboretum and ornamental plantings of Emerald Caroudel, (an interspecific hybrid between B. koreana and Berberis Figure 2. The general procedure adopted for the identification of aecial hosts of Puccinia striiformis f. sp. tritici (Pst) under controlled conditions. A; Growing barberry plants in the greenhouse using seeds of different barberry species. B; 10-15 seeds are placed on the blotter paper in the Petri-plates and moistened with distilled water. The Petri-plates are placed in the incubator at 10°C, 12/12 h dark/light, and 100% relative humidity (RH). Every next day the seeds are moistened with distilled water using a hand button spray machine. C; at 2-3 leaf stage, the barberry seedlings are transferred into plastic trays and incubated at the same conditions. D; at 3-4 leaf stage, the seedlings are transferred into the earthen-pots, covered with plastic cylinders, and transferred into a growth room having 22-25°C and 60% RH. E; a collection of urediniospores of a dominant race of Pst. F; isolation of a single urediniospore isolate. G; mass multiplication of single urediniospore isolate. H; inoculation of adult wheat-plant (at the growth stage 50) for the production of teliospores. I; teliospore germination and artificial inoculation of young leaves of barberry plants with basidiospores and checking completion of pycniospores on the upper surface of leaves and aeciospores on the lower surface under controlled conditions. J and K; inoculation of 10-day-seedlings of a susceptible wheat variety with aeciospores from the aecial cups. Observation of typical urediniospores usually 10-18 days after inoculation. (also known as European barberry or common barberry), is the most common barberry which is widely grown in central Asia, North Africa, Europe, and the Middle East, and was introduced to North America from Europe. A representative shrub of barberry (Berberis pseudumbellata), showing inflorescence and shoots with ripened berries, naturally growing in the Himalayan region of Pakistan is shown in Figure 3. Up till now, a total fifty-three Berberis and Mahonia species susceptible to Pst, under controlled and natural conditions, have been identified by different researchers. The historical perspectives for the identification of alternate hosts of the wheat stripe rust pathogen are given in Table 5.

Disease epidemics and pathogen diversity
Alternate hosts of the wheat stripe rust pathogen are considered to play an essential role in over-summering the pathogen in adverse environmental conditions and offer new inoculum (generated through sexual recombination) for the development of the wheat stripe rust disease epidemics. According to Zhao et al. (2016a), the function of an alternate host of cereal rusts depends upon fungal species and ecological conditions for the disease epidemic and pathogenic diversity. Generally, an alternate host provides survival to the pathogen during adverse conditions serving as a sexual host and may generate diversified pathogenic populations through sexual recombination (Jin 2011;Zhao et al. 2016b).
The alternate hosts of Pst are known, but their specific role in the underlying mechanism of virulence diversity, phenotypic and genotypic changes and sexual recombination is not yet fully explored. The discovery of barberry as aecial host of the wheat stripe rust pathogen suggests an essential role

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of sexual reproduction in the genetic diversity of the pathogen. However, the inherent part of barberry species in sexual recombination causing genetic diversity is still unclear. Earlier studies (Roelfs 1982;Burdon and Roelfs 1985) investigated the role of alternate hosts in generating virulence diversity. Environmental conditions, including weather conditions and cropping systems, are highly crucial for the wheat stripe rust disease epidemics. The environmental conditions conducive for all rusts are specific. The disease development by urediniospores or aeciospores on cereal crops, and by basidiospores on alternate hosts require special requirements of temperature and humidity. The environmental conditions not only effect on survival, infection, growth, and reproduction of the fungi throughout the asexual cycle but also affect the different stages of the sexual cycle. For example, the survival of teliospores, their germination, and basidiospore infection on alternate hosts (Rapilly 1979 Considering their function in the survival of the Pst spores (teliospores, basidiospores, aeciospores) during adverse environmental conditions. For example, wheat is largely grown as a winter crop in the valleys of the Himalayan region in Pakistan to fulfill the requirements for human food and

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animal feed. In the lower parts (1,200-1,900 m above sea level) of valleys, a double-cropping system is used for growing two staple crops, wheat from November to May and maize from June to October. In the areas of high altitudes (2,300-3,000 m above sea level), maize is the main crop grown from May to October or June to November, and summersown wheat is grown as a secondary or minor crop (Hashmi and Shafiullah 2003;Ali et al. 2014a). In this region, both primary and alternate hosts coexist, an ecological condition required for Pst to complete the macrocyclic lifecycle on these distinct plants. Jin et al. (2010) hypothesized that in areas where wheat and stripe rust-susceptible Berberis spp. coexist, sexual recombination likely plays an active role in contributing to the diversity of Pst. Ali et al. (2014b) proposed a sink and source relationship between non-Berberis and Berberis zones for the significance of the sexual cycle of Pst populations in the center of origin. Pst basidiospores could potentially infect barberry spp. in the Himalayan region if the teliospores on wheat plants can germinate in the spring or late in the fall and remain throughout the dry winter as reported in Gansu, China (Zhao et al. 2013;. However, whether barberry plants provide aeciospores to start the wheat stripe rust disease in wheat crops in the Himalayan region needs further studies. Identification of aecia on naturally infected barberry plants infecting wheat plants and using molecular markers , is the most direct and powerful approach to answer the questions whether and to what extent alternate hosts are important for the wheat stripe rust disease epidemics in the Himalayan region of Pakistan and other regions in the world. High genetic diversity for both virulence and molecular markers were discovered in P. striiformis populations in the Chinese Gansu province and in the Middle East (Bahri et al. 2009;Mboup et al. 2009;Duan et al. 2010) which emphasize the hypothesis of periodic recombination in these regions, even though the exact mechanisms were not determined. However, the isolates from China more readily produced telia than the isolates obtained from Europe, suggesting that the tremendous diversity in Gansu is due to sexual reproduction (Ali et al. 2010). The results of these observations are in contrast to several studies in Europe (Hovmøller et al. 2002;Enjalbert et al. 2005), Australia (Steele et al. 2001), and the Yunnan area of China (Liu et al. 2011), where the genetic diversity was generally low and consistent with a clonal Pst population structure. These results show that the role of sexual recombination in Pst is different among regions, and depends on the opportunity for sexual reproduction and somatic hybridization. However, the details and the impact of both processes under natural conditions remain to be investigated.

Dynamics of alternate hosts
The functions of alternate hosts are greately influenced by several factors. For example, the taxonomic entities of susceptible alternate hosts are essential because these serve not only as a carrier but also as a breeder of possible new and aggressive strains of the the wheat stripe rust pathogen. The most important thing is to distinguish barberry species without knowing their cultural and genetic origin. Different authors may have given plants with the same morphology and physiology different names to one barberry spp. Several questions are also related to the taxonomy of the barberry plants.
Protocols for the identification of different barberry spp. (such as photos, drawings, previous literature, and webbased data) can be helpful for taxonomical identification of barberry species in an area. However, the taxonomic classification of different barberry species is still controversial in some areas in the world (e.g. the Himalayan region in Pakistan), and it needs further investigations, preferably DNA based technology to classify the barberry species/subspecies (Khan et al. 2015).
Another important factor is the phenology of alternate hosts and ecological surroundings, essential for Pst pathogen to infect primary and alternate hosts in a specific area (Mehmood et al. 2019). Wang and Chen (2015b) reported that teliospores were produced on winter wheat (from June to July) and on spring wheat (from July to August) in the US Pacific Northwest. Under controlled conditions, the mature teliospores freshly harvested from wheat leaves can germinate readily. The germination rate will decrease over time. The annual precipitation in the region occurs mainly during the winter from November to February. From middle April to early May, new leaves of barberry emerge, but at that time, teliospores are not viable, resulting in the absence of aecial infection. While teliospores of P. graminis f. sp. tritici (Pgt) are produced from June to July, remain dormant until the winter ends. They start germinating in April to May and infect young leaves of susceptible barberry plants to cause infection in May and June. As a result of this infection, aeciospores are released to infect cereal crops in June and July. Therefore, barberry in this region plays a vital role in Pgt but not for Pst Wang and Chen 2015b).

The important roles of the alternate hosts
One important role of alternate hosts of the wheat stripe rust pathogen is the production of new aggressive races with combined virulence genes through sexual reproduction as described in case of P. graminis f. sp. tritici (Pgt) (Stakman et al. 1934) P. graminis f. sp. avenae (Pga) (Newton and Johnson 1944) P. coronata f. sp. avenae (Wahl et al. 1960) indicating that alternate hosts increase the number of races (Carson 2008(Carson , 2011. Similarly,  identified 10 races of Pgt from 16 single-uredinium cultures from aecia on barberry bushes in northern Idaho in the US. Another important role of the alternate hosts of the wheat stripe rust pathogen is population diversification through sexual reproduction on its alternate hosts e.g. barberry. For example, the number of new races was significantly larger in the areas with alternate hosts than the barberry eradication regions in the case of Pgt. A comparison of virulence phenotypes of Pgt (Roelfs and Groth 1980) near barberry plants in eastern Washington and northern Idaho (reproduced sexually) and of populations east of the Rocky Mountains (reproduced asexually) resulted in the identification of 100 races (23.5%) out of 426 isolates from the sexual populations, and 17 races (0.07%) out of 2,377 isolates from asexual populations. Similarly in the US Pacific Northwest, high genetic diversity in the Pgt population has been observed in recent years (Jin et al. 2014;Wang and Chen 2015b). High genetic diversity has also been reported for a sexually produced P. cerealis population (Al-Kherb et al. 1987). Although a large number of rather rare races collected from alternate hosts, or cereal and grasses near alternate host plants have been reported for Pgt, indicating the importance of alternate hosts in generating diverse races, however a lot of investigations are needed in case of the wheat stripe rust pathogen especially from the Pst infected barberry plants under natural conditions.

Research progress; revealing the genetic diversity of Pst
Pst genetics has been studied using the alternate host plants especially the Berberis spp. But most of the investigations about the avirulence and virulences are done under greenhouse conditions. The pathogenicity characters or traits like latency, lesion size, spore production rate, sporulation duration, spore color, telial formation have also been considered (Zhao et al. 2016a(Zhao et al. , 2016b. The most common approach to access and quantify the genetic factor involved in pathogenic diversity is to measure the value of concordance information obtained from the individuals of a population. Researchers have utilized common barberry species as a model system for the genetic studies of Pst. Recently barberry has gained much concern in Pst studies due to the increased interest in understating the genetics of the pathogen. The barberry plants serve as alternate hosts for Pst, and sexual populations can only be generated on barberry under controlled conditions. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. A 110-Mb draft sequence of a Pst isolate CY32 was reported by Zheng et al. (2013) using 'fosmid-to-fosmid' strategy. Re-sequencing analyses showed a high genetic diversity of six Pst isolates collected from different continents. The draft genome assembly, in association with transcriptomics, provides the first insight into the molecular biology of Pst. A large number of Pst populations of a diverse geographical origin were analyzed using microsatellite markers or simple sequence repeat (SSR) markers. The results showed a higher genotypic diversity, recombinant population structure, and high sexual reproduction ability in the Himalayan and neighboring regions (Nepal, Pakistan, and China) suggested this area as the putative center of origin of Pst (Ali et al. 2014a). The virulence phenotyping and molecular genotyping approaches were used to study the genetic diversity of PST populations in three epidemiological regions in China, and the results revealed that the Xinjiang region had a higher genetic diversity of Pst as compared to other epidemic regions . However, these studies lack detailed sequence information of fundamental genetic changes in pathogen populations. Whole-genomic and transcriptomic data into field pathogen surveys can help to understand this fundamental question (Hubbard et al. 2015). It is easy to identify causative alleles of DNA sequence variations and changes in pathogen fitness using potential genomeguided techniques (Schwessinger 2016).
The application of various molecular markers, such as amplified fragment length polymorphism (AFLP), fragment length polymorphism (FLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR) have been used to understand the genetic diversity of Pst (Tian et al. 2016;Yuan et al. 2017). The whole-genome re-sequencing of several isolates offers an ideal resource for the development of SSR markers, which are useful for genetic and population studies of Pst (Luo et al. 2015). The single nucleotide polymorphism (SNP) has been used as the standard genetic marker to identify disease-associated alleles. Using SNP genotyping technology, we can efficiently investigate genotype variation across 100 000-1 000 000 SNPs. Quantitative trait locus (QTL) on DNA correlates with variation of the quantitative trait in the phenotype in the progeny population of Pst. The goal of this process is to identify the action, interaction, number, and precise location of these regions. It identifies which molecular markers (such as SSRs, AFLPs or SNPs) correlate with an observed trait. Molecular techniques have greatly increased the available markers in rust fungi for constructing linkage maps (Zhao et al. 2016a(Zhao et al. , 2016b. Due to the evolution of new virulent races, the resistant cultivars become susceptible after a few years in a particular region (Chen 2005). Every year new races of Pst are identified in wheat-growing areas in the world, especially in the US, China, India, Pakistan, and other countries (Chen 2005;Zhan et al. 2013;Wan and Chen 2014;Wan et al. 2016). The mechanism involved in the virulence diversity and genetic variation is essential to understand the role of alternate hosts of the wheat stripe rust pathogen. Few studies have been reported on the genetics of Pst virulence. The first study of the genetics of Pst demonstrating virulence and avirulence genes and gene-for-gene relationship in the Pst-pathosystem was conducted by Wang et al. (2012). They selfed a US isolate of race Pst-127 on B. vulgaris and obtained 29 progeny isolates. They tested the isolates on Yr single-gene lines and found parental isolate as homokaryotic (homozygous) for virulent loci to Yr1, Yr2, and Yr9 and for avirulence loci to Yr5, Yr15, Yr24, Yr32, and YrSP. In contrast, its segregation was observed for virulence phenotypes (VPs) to Yr6, Yr7, Yr8, Yr10, Yr17, Yr19, Yr27, Yr43, Yr44, YrExp1, YrExp2, YrTr1, and Yr76 (YrTye) in different ratios. The avirulence to seven Yr genes (Yr6, Yr7, Yr8, Yr19, YrExp2, and YrTye) was dominant and controlled by single genes in the parental isolate. In contrast, avirulence to Yr17 and YrExp1 in the parental isolate was controlled by individual recessive genes. Avirulence to Yr44 was controlled by two independent dominant genes and avirulence to Yr43, and an unknown Yr gene was controlled by two recessive genes (7:9 ratios). Tian et al. (2016), selfed a Chinese Pst isolate (Pinglan 17-7) on B. shensiana and obtained 118 progeny isolates. They found 24 VPs, 82 MLGs using 13 polymorphic SSR markers. A preliminary linkage map was constructed with 8 of 24 avirulence/virulence loci and 10 SSR markers. Avirulence to Yr4, Yr32, and Yr44 in the parental isolate was controlled by two recessive complementary genes (1:15 ratios). A preliminary linkage map was constructed with 8 of 24 avirulence/ virulence loci and 10 SSR markers. They found that a highly diversified genetic population of Pst can be generated by selfing a single isolate on barberry, and this progeny population can be used for its virulence characterization. In another study using the same method, 120 progeny isolates were obtained by selfing another Chinese isolate. They tested them on 25 Yr single-gene lines and found 51 VPs and 55 MLGs using 11 polymorphic SSR markers. Another linkage map was constructed using four avirulence loci and 11 SSR markers (Tian et al. 2017). Rodriguez-Algaba et al. (2014) studied the genetic diversity within and among aecia of Pst produced on B. vulgaris. The genetic markers confirmed segregation and resulted that the progeny isolates were derived from the parental isolate through sexual reproduction. Mehmood (2017), generated a segregating population of 119 isolates by self-fertilization of P. striiformis f. sp. tritici isolate 08-220 (race PSTv-11) on barberry leaves under controlled greenhouse conditions. They constructed a genetic map of six linkage groups using a massive amount of genotype-by-sequencing (GBS) SNP markers. All these studies reported the dominance and recessiveness of avirulence or virulence genes. These results may help to understand the genetic diversity of Pst, the role of sexual reproduction, host-pathogen interaction, and selection of resistance genes in breeding programs. In future the use of several novel technologies such as auto-fluorescent proteins in combination with confocal laser scanning microscopy (CLSM), single-molecule detection, atomic force microscopy, and differential fluorescence induction (DFI) combined with optical trapping (OT) (Allaway et al. 2001) are useful to study microbial behavior of Pst.

Prospects
The most promising future tasks to understand the genetics of Pst and the role of alternate hosts are: a) To study proteins involved in the mechanism of diversity in the progeny population either through sexual recombination or clonal reproduction. b) To study the extent to which biotic and abiotic environmental conditions cause variation in virulence diversity of Pst through sexual or asexual reproduction. c) To identify Pst effector proteins and their roles in the pathogenesis and alternate host susceptibility under controlled and natural conditions. d) To understand the diversity mechanism in Pst and the evolution of these devastating races of the pathogen through the use of molecular techniques. e) To analyze the genomes of dominant virulent strains of Pst that caused huge wheat yield losses worldwide. f) To compare the complements of avirulence/virulence effector genes from different Pst genomes using bioinformatics techniques. g) To use newly generated high-density genomic data of Pst pathogen to assess virulence diversity.

Conclusion
Although several barberries and Mahonia spp. have been known as susceptible hosts to Pst however Berberis and Mahonia represent several diverse sections, and more susceptible hosts are likely to be identified in the future. Alternate hosts of Pst help the pathogen survive adversarial environmental conditions and provide new inoculum through sexual reproduction to infect wheat, other cereals, and grasses. They are essential facilitators to Pst, and they provide shelter for the survival of the pathogen; provide initial inoculum for the disease epidemics under favorable conditions; diversify pathogen populations through sexual reproduction, and may cause the emergence of virulent races of the pathogen. Phenology of alternate hosts and ecological conditions also play an essential role during the life cycle of Pst. It is needed to study the molecular mechanism involved in pathogenicity, Pst effector proteins, and susceptibility of the alternate hosts under natural conditions. Computational estimation is that Pst genomes encode over 1000 candidate effectors (Cantu et al. 2013;Zheng et al. 2013). In the future, we need to know about the genetic frequencies, identification of effectors within Pst populations to encompass wheat stripe rust durably (Ellis et al. 2014). Regardless of the importance of alternate hosts, the most important thing is to cultivate resistant cultivars to control Pst through the identification of both resistant genes of wheat cultivars and virulent races of the pathogen. Due to the emergence of new races of Pst, a rapidly evolving threat to global food security, dramatic and urgent actions are needed at all levels throughout the wheat-growing regions in the world, to limit the spread of Pst. It is essential to emphasize on the Urgency of International Cooperation, in several dimensions, rapid, accurate, and transparent international communication about the unfolding epidemiology of the wheat stripe rust disease, including patterns of spore dissemination, its origins, genetics, and mutations either clonal or sexual, and strong coordination in all activities in these areas. Sharing of information in the research and development along with collaborative research efforts to deal with Pst. Mainly focus on the study of the interaction between phytopathogenic fungi and wheat, explores systematically on wheat disease resistance mechanism and pathogenic mechanism from such aspects as histology, cytology, molecular cytology and molecular biology and formed a characteristic research system.