Characterization of wheat-Thinopyrum bessarabicum genetic stock for stripe rust and Karnal bunt resistance

Abstract Utilization of modern breeding techniques for developing high yielding and uniform plant types ultimately narrowing the genetic makeup of most crops. Narrowed genetic makeup of these crops has made them vulnerable towards disease and insect epidemics. For sustainable crop production, genetic variability of these crops must be broadened against various biotic and abiotic stresses. One of the ways to widen genetic configuration of these crops is to identify novel additional sources of durable resistance. In this regard crops wild relatives are providing valuable sources of allelic diversity towards various biotic, abiotic stress tolerance and quality components. For incorporating novel variability from wild relative’s wide hybridization technique has become a promising breeding method. For this purpose, wheat-Th. bessarabicum amphiploid, addition and translocation lines have been screened in field and screen house conditions to get novel sources of yellow rust and Karnal bunt resistant. Stripe rust screening under field conditions has revealed addition lines 4JJ and 6JJ as resistant to moderately resistant while addition lines 3JJ, 5JJ, 7JJ and translocation lines Tr-3, Tr-6 as moderately resistant wheat-Thinopyrum-bessarabicum genetic stock. Karnal bunt screening depicted addition lines 5JJ and 4JJ as highly resistant genetic stock. These genetic stocks may be used to introgression novel stripe rust and Karnal bunt resistance from the tertiary gene pool into susceptible wheat backgrounds.

Haryana, India in 1931 (Mitra 1931).Bonde et al. (1997) and Rush et al. (2005) have also reported the prevalence of this disease in other countries.Karnal Bunt infection does not cover the entire wheat ear, rather it restricts its infection to a few kernels within a spike and to a part of the grain and rarely the whole grain, thereby, the disease has also been given the name partial bunt (Pandey et al., 2019).The fungus infests the ovaries in the emerging wheat heads resulting in partially or completely filled powdery masses of teliospores emitting a foul smell of trimethylamine (Shakoor et al., 2015).Wheat grains infected with KB are of low quality as they port an unacceptable odor, color, and taste and at as low as 1% infection the grains/flour become unpalatable (Kashyap et al., 2018).Most devastating effect of Karnal bunt (KB) is to quarantine regulations that may restrict the free global trade of wheat besides the loss of grain yield and quality as well (Bishnoi et al., 2020;Sukhwinder et al., 2007).Cultural practices and chemical treatments have been futile due to soil-borne and an airborne sporidial stage (Carris et al., 2006;Garrett andBowden, 2002, Kumar et al., 2014).The morphological barriers as well as the physiological traits manifests the genetic resistance against KB fungus in wheat and Durum.For example, the higher degree of resistance expressed by triticale and durum wheat in comparison to bread wheat is attributed to the morphological defense barriers like pubescence rather than it being physiological (Warham, 1988).The morphological traits like leaf sheath, flag leaf base, glumes and rachis with higher number of stomata and glumes and rachis with low hair count and compact arrangement of spikelets are relevant to the resistant lines (Bishnoi et al., 2020).The Gogoi et al. (2002) elaborated that relatively more resistant durum wheat and triticale have lower glume opening and low ear emergence period.Thus, the best approach to protect the crop from yellow rust and Karnal bunt pathogens is to breed for genetic resistance.(McIntosh et al., 2018).Due to limited genetic diversity within cultivated crops (Reif et al., 2005), cytogeneticists, breeders and farmers are sorting out additional and novel sources of resistance from primary, secondary, and tertiary gene pools of wheat (Feuillet et al., 2008, Milus et al.., 2015).Wheat wild relatives from tertiary gene pool are providing valuable sources of genetic diversity having a variety of R genes for rust and Karnal bunt resistance that could enable more sustainable disease control (Kerber and Dyck, 1990;Narang et al., 2020).Genes from over 52 allien species including T. umbellulatum, T. comosum, Thinopyrum intermedium, Th. elongatum, Th. ponticum and Secale cereale have been introgressed (McIntosh et al., 1995) into wheat to exploit natural variation of alien species for wheat improvement (Wulff and Moscou, 2014).
In this regard, characterization of various wheat-allien genetic stocks for identification of such novel resistances is paramount.Keeping in view the importance of Yellow rust and Karnal bunt diseases, Present study was carried out to screen wheat-Th.bessarabicum genetic stocks possessing resistance against stripe rust and Karnal bunt to enhance cultivar improvement by using these wheat-allien genetic stocks in breeding programs in Pakistan.

Introduction
World's diet constitutes about 93 percent of the plants.Among these plants, cereals contribute two-thirds of all food.Wheat, maize and rice constitute about 80 percent of total global cereal production (Getachew and Biruk, 2018).Wheat (Triticum aestivum L.) is the world's leading cereal grain used as staple food by more than one third of the world's population (FAO, 2018) .It is a leading source of calories as well as protein consumption for both humans and livestock (Narang et al., 2020).Modern day agriculture is facing severe challenges from biotic and abiotic stresses, threating its food security and sustainable development (Jinya et al., 2020).Among various kinds of biotic stresses, fungi are the most devastating one causing the majority of plant diseases (Robert-Seilaniantz et al., 2011).Wheat crop is hosted by variety of fungal pathogens which causes infection at different developmental stages; among them the important ones are stripe rust and karnal bunt.(Bishnoi et al., 2020).Most important wheat foliar disease yellow rust (stripe rust) is caused by a fungus known as Puccinia striiformis f. sp.tritici.Almost all the continents except Antarctica host this disease (Ali et al., 2017).Wheat grain loss of about 30 to 100% due to this fungus imposing global threat to wheat production (Chen, 2005).Various reports have revealed that this disease had the ability to destroy the entire wheat crops under favorable conditions (Mengesha, 2020).In Pakistan, losses due to this disease are not estimated (Shakoor et al., 2015), but at global level yield loss of at least 5.5 million tons per year is caused by yellow rust disease (Marcelo et al., 2020).Puccinia striiformis f. sp.destroys the plants respiratory system by causing necrosis, stunting plant growth and by reducing grain yield (Line, 2002;Chen, 2005).Defending measures against the destruction of this pathogen are of primary concern for world food security (Chakravarty, 2011).Diverse disease control strategies like use of chemicals and agronomic practices are proven profitable to lessen the losses (Foster et al., 2017), but it has been reported that recurrent use of the same chemical fungicide for several years under extensive wheat production area might favor the development of resistant pathogens ultimately necessitating the use of alternate fungicide (Getachew and Biruk, 2018).So, in this situation host genetic resistance is the most practicable method to control stripe rust alternatively.Crops genetic resistance exploitation is economical, and bears no health and environmental hazards and proving resistant for a longer period of time ensuring crop sustainability (Hovmøller et al., 2017;Farrokhi et al., 2011).Stripe rust resistance of two types, all-stage resistance (also called race specific) and adult-plant resistance (also called race nonspecific) have been identified.Among these two types, adult plant resistance (Apr) has been found to be effective against all races conferring durable resistance.(Chen, 2013).On the contrary, race specific resistance becomes ineffective within three to five years (Line and Qayoum, 1992).Strategy of pyramiding these major (race specific) and minor (race non-specific) genes (Singh et al., 2004)

Field evaluation for stripe rust resistance
Wheat-Th.bessarabicum genetic stocks along with Chinese Spring wheat variety, and standard check variety Morocco (Table 1) were screened for adult plant resistance in field conditions of NARC Islamabad during wheat growing cycles (2012)(2013)(2014)(2015).The material was raised in Randomize Complete Block design (RCBD) with three replications.Each plot consisted of one row of 2.5 m length spaced at 30 cm between rows and 15 cm between plants.Every fifth row was seeded with Morocco check cultivar.Other recommended cultural practices for wheat production were followed during the growing seasons.All the genetic stock was inoculated thrice by Uredio-spore suspension to create stripe rust epidemic after 6 weeks of planting.The data were analyzed by using modified Cobb scale (Mengesha, 2020).

Disease scoring for adult plant resistance
Data was taken at flag leaf stage for disease severity and infection after every 10-days interval.The data was recorded by using modified Cobb scale (Mengesha, 2020) as percent (%) of the rust infection on the plants surface.Field response referring to the type of infection was recorded according to the Table 2.The data was recorded for disease severity first time when the susceptible check (Morocco) had reached up to 100% disease infection (Table 2).

Karnal bunt 2.3.1. Inoculation technique and disease scoring
For Karnal bunt screening, 1 ml tiller -1 sporodial suspension taken from Crop disease research institute (CDRI) of NARC Islamabad was injected by using a hypodermal syringe at booting stage (Stages 48-49) (Zadoks et al., 1974) for two consecutive wheat growing cycles in NARC fields, i.e., 2013-2015.Tagging of injected tillers was done.Susceptibility category was evaluated for all genetic stocks inoculated on the basis of CI Table 3, following the susceptibility category given by Aujla et al. (1989).

Discussion
Wheat is the largest cereals crop of the world.Despite its importance as cereal crop, its yield and productions are prone to various biotic and abiotic factors among which wheat rust diseases are the most important.One of the most important objectives of wheat breeding programs in all wheat growing regions of the world is to develop durable tolerance against yellow rust in wheat cultivars (Akfirat et al., 2010).Durable resistance can be obtained by pyramiding various ASR and APR genes into one variety (Klarquist et al., 2016).Due to swift breakdown of commercially deployed resistance genes, characterization of diverse and novel sources of resistance is constantly needed to replace the defeated genes.Wheat wild relatives are a potential source of novel rust resistance genes for developing new and diverse resistant germplasm (Kerber and Dyck, 1990).In present study, wheat-Th.bessarabicum addition line 4JJ and 6JJ exhibited resistance attitude towards yellow rust (Table 4).As these lines were not immune to yellow rust indicating the presence of minor genes which are desirable for durable resistance.Two novel QTLs for adult plant resistance have already been identified on group 4A and 6B chromosomes of wheat (Klarquist et al., 2016).Other addition lines 3JJ, 5JJ, 7JJ, and translocation lines Tr-3 (3JS.3BL),Tr-6 (6JS.7DL) were ranked as moderately resistant.Several QTLs for stripe rust resistance have been shown in group 3, 5 and 7 of wheat (Rosewarne, et al., 2013).It can be concluded that chromosome 3J, 5J, 4J, 6J and 7J of Th. bessarabicum may possess some of the adult plant resistance (APR) genes loci which have provided adult plant resistance against stripe rust in 3JJ, 5JJ, 4JJ, 6JJ and 7JJ additions and translocation lines in CS background.Data for CS and amphiploid could not be taken due to late maturity of these materials.Karnal bunt or partial bunt, caused by T. indica (syn.Neovossia indica [Mitra] Mundkur) also occurs endemically in (Punjab) Pakistan (Sajjad et al., 2018).For screening the germplasm, boot inoculation technique is one of the useful methodologies that allow the maximum ratio of successful infection (Beniwal et al., 2001).There is scarceness of resistance in the commercial cultivars against Karnal bunt in the country (Raza et al., 2019) and across the border (Bishnoi et al., 2020).Due to lack of resistance in commercial cultivars (Shakoor et al., 2015) wheat Th. bessarabicum genetic stock has been evaluated by boot inoculation technique.Results showed that amphiploid and addition lines 4JJ, 5JJ are immune to the KB while addition line 2JJ, 3JJ, 7JJ, translocation line , and two BC1s self-fertile lines were ranked as resistant (Table 6).KB resistance in wheat is polygenic (Brar et al., 2018;Gupta et al., 2019).This polygenic resistant attitude was partly based on the fact that six wheat chromosomes (1D, 2A, 3B, 3D, 5D, 7A) were attributed to influence the reaction against the pathogen (Gill et al., 1993;Singh et al., 1994). .In 2019, Gupta et al. also reported novel QTLs on chromosomes 1DL, 2DL, 4AL, 5AS, 6BL, 6BS, 7BS, and 7DL.Nine other QTLs were also detected on chromosomes 3B, 4A, 4B, 5A, 5B, and 7A (Brar et al., 2018;Singh et al., 2007).It is probable that there are numeral genes that affect resistance against KB, because diverse mechanisms could operate for shielding the plant against the pathogen and each of them may be controlled by different genes (kumar et al., 2019;Gurjar et al., 2019;  20.0 and above Highly Susceptible (HS) (Aujla et al., 1989).
Brazilian Journal of Biology, 2023, vol.83, e246440 5/8 Wheat-Thinopyrum bessarabicum genetic stock screening for stripe rust and karnal bunt  Emebiri et al., 2019).This defense mechanism possesses phenotypic barriers (wax, cell wall, stomatal aperture or lenticles) and chemicals comprise of a diverse array of secondary metabolites (phenolics, sulphur compounds, saponins, cyanogenic glycosides, and glucosinolates) have been synthesized by plant, many of which are deterrent to fungal activity (Osbourn, 1996).Resistance due to secondary metabolite has also been observed in Rice blast disease against different strains of Magnaporthe oryzae (Singh et al., 2020).Broad-spectrum resistance has also been observed due to resistance (R) and defenseregulator(DR) genes to the blast disease of rice (Li et al., 2019).In the light of these studies, it can be demonstrated that amphiploid and addition lines 4JJ and 5JJ might possess genes encode various plant defense mechanisms including some of above-mentioned physical barriers and or chemical barriers scattered on 4J and 5J homoeologous chromosomes from Thinopyrum bessarabicum which confer immunity against KB.This result is in accordance with the finding that QTLs for KB resistance are present on 5D (Begum and Mathur, 1989) and on 4B (Sukhwinder-Singh et al., 2003;Emebiri et al., 2019) chromosomes of wheat.Resistance against KB has also been found in the screening of wheat and rye addition lines on 4R, 5R and long arm of 7R chromosomes (Sidhu et al., 2001).Other addition line 2JJ, 3JJ, 7JJ, translocation line Tr-1 (6BS.6BL-6J),Tr-3 (3JS.3BL),Tr-5 (7DS.7DL-4J),Tr-7 (5JS-5DS.5DL),, and two BC1s self-fertile lines ((2n=7X=42+7J) also possess resistance gene(s) distributed on different homoeologous Th. bessarabicum chromosomes/ chromosomal arms, as QTLs for KB resistance are also present on 2A, 3B, 3D, 5D, 7A (Begum and Mathur, 1989;Emebiri et al., 2019) and 4B chromosomes of wheat (Sukhwinder-Singh et al., 2003).Homoeology of Thinopyrum bessarabicum chromosomes with wheat have already been explained in another study carried out by William and Mujeeb-Kazi (1995).These highly resistant addition and translocation lines of wheat-Th.bessarabicum identified in the present screening stands as a preferred candidate for KB resistance as an added positive attribute from tertiary gene pool.

Table 2 .
Rust reaction, infection type for field response and response value.

Table 3 .
Standardization of vulnerability category based on CI value.

Table 5 .
Susceptibility category of each wheat-Thinopyrum bessarabicum genetic stock, based on coefficient of infection when inoculated with mixture of isolates.