Genetic Enhancement and Genetic Analysis of Resistance to Asian Soybean Rust in India

Asian Soybean rust (ASR) is the most threatening fungal disease in the major soybean production areas around the world. ASR is an economically important disease due to the formidable threat it poses to the world’s soybean crop. The causal agent of ASR is Phakopsora pachyrhizi Syd., an obligate biotrophic fungus. First identified in Japan in 1902, the pathogen has since been observed in all major soybean-growing regions of the world and has caused substantial economic damage, losses up to 10-90% (Sarbhay and Pal, 1997) to regions of Asia and South America over the past two decades. The fungus has several races with multiple virulence alleles which suggest that the ASR pathogen has high genetic variability. Currently, chemical spray containing fungicides is the only effective method to control the disease. In spite of the availability of chemical control with fungicides, the increase of production costs as well as operational difficulties associated with fungicide use for disease management has prompted the search for to development of durable resistance to Phakopsorapachyrhizi. Attempts to develop cultivars that are resistant to SBR have been undertaken around the world [India (Singh et al., 1975), Taiwan (Wang and Hartman 1992) and Nigeria (Twizeyimana et al., 2008)] and SBR resistant cultivars with specific genes have been released in Uganda (Oloka et al., 2008). To date, however, no resistant cultivars appear to have been cultivated on a large scale in these countries.
       
Information on the host differential response and genetics of resistance has led to identification of six different rust resistant genes (Rpp: Resistance to P. pachyrhizi), viz., Rpp1 to Rpp6, against specific isolates of P. pachyrhizi (Hartman et al., 2005; Bonde et al., 2006; Miles et al., 2011). Rpp1 confers an immune response for which there are novisible symptoms in the plant (Miles et al., 2006). Resistance responses mediated by the Rpp2 to Rpp5 loci results in the formation of visible reddish-brown lesions which limit fungal growth and sporulation, thereby suggesting of a hypersensitive-like response (Bonde et al., 2006; Garcia et al., 2008). Rpp6 was relatively resistant to rust in most soybean producing areas worldwide, although some susceptible reaction was evident (Kato, 2017). The susceptible interaction with rust results in tan colored lesions and fully sporulatinguredenia (Bromfield and Hartwig 1980; Bromfield, 1984; Miles et al., 2006).
       
This differential reaction of specific cultivars to specific races of pathogen raises the need for further investigation of the genetics of resistance to soybean rust. The genetic characterization of disease resistance in plants is essential for the understanding of plant pathogen interactions. These concepts contribute to an efficient breeding programme for disease resistance.
       
Soybean rust take maximum toll in Southern India causing significant yield losses. The disease is spreading fast to new areas throughout India and in particular southern states such as Karnataka, Maharashtra and Andhra Pradesh, besides others. Indeed, the fast spread of rust disease has become a major concern for expanding area under soybean in South India. Breeding for resistant varieties with greater yield stability and discovery of single-gene or partial resistance will be an economically effective approach for long-term rust management (Hartman et al., 2005).
       
Most of the popular soybean cultivars cultivated in India are susceptible to rust and therefore a breeding programme was initiated at the University of Agricultural Sciences, Dharwad, India to develop rust resistant genotypes. After rigorous screening of more than 2000 germplasm lines, two rust resistant germplasm lines viz., EC 241778 and EC 241780 were identified. JS 335 is the most widely cultivated soybean variety whose popularity lies in its high yield, early maturity and high adaptability in soybean cultivation regions of India. However the variety is highly susceptible to rust disease which causes serious yield losses. Utilizing JS 335 and EC 241778 and EC 241780, in a long term breeding programme, a highly rust resistant variety, DSb 21 has been released from University of Agricultural Sciences Dharwad, Karnataka. This is the first rust resistant variety released for Southern Zone of India.
       
Further, the study was aimed at genetic analysis of highly resistant exotic germplasm line (EC 241778) and new rust resistant variety (DSb 21) in order to provide a scientific basis for applying in soybean production. 

A long term breeding programme was initiated during 1998 at the University of Agricultural Sciences, Dharwad through germplasm evaluation, induced mutations and hybridization (pedigree/backcross method) for the development of rust resistant genotypes with high yield potentiality. The details of the breeding procedure followed is schematically represented in Fig 1a and 1b.
 

 

       
About 2,200 germplasm lines used in the present study were procured from different sources viz., AVRDC, Taiwan; Indian Institute of Soybean Research, Indore; NBPGR, New Delhi; University of Agricultural Sciences, Bangalore; JNKVV, Jabalpur and accessions maintained at  AICRP on Soybean, University of Agricultural Sciences, Dharwad, India. These lines were screened against rust during rainy seasons of 2000 to 2003 at the Main Agricultural Research Station, University of Agricultural Sciences, Dharwad and R and D Farm, Ugar Sugar Works, Ugarkhurd, the hot spots for rust disease in India. The identified rust resistant exotic germplasm lines, EC 241778 and EC 241780 were utilized in hybridization programme with agronomically superior but rust susceptible cultivars viz., JS 335, JS 93-05 and DSb-1 during the year 2005. The salient features of the parental lines used in the study are given in Table 1. Lot of segregating material was generated using these parental lines. More than 500 advanced breeding lines were selected for preliminary screening under natural field condition and about 182 lines were finally selected. These 182 advanced breeding lines were screened under natural epiphytotic conditions as well as artificially inoculated glass house condition for two years during 2007 and 2008. The screening techniques were followed as suggested by Shanmuga sundram (1977) and Mayee and Datar (1986). Among them, 11 lines exhibited resistant reaction under glass house condition and varied reaction (MR to HR) under natural epiphytotic conditions at Dharwad (Table 2a). Further evaluation of these 11 lines lead to the identification of three promising rust resistant lines during 2009.  These three lines viz.,  DSb 21 (JS 335 × EC 241778), DSb 23-2 (JS 335 × EC 241780) and DSb 28-3 (JS 93-05 × EC 241780) were further evaluated for their reaction to rust and yield potentiality under rust prone conditions at hot spots viz., Ugarkhurd and Dharwad during 2010 to 2013 in randomised block design, with three replications and spacing of 30 × 10 cm. Based on the above study, DSb 21 was released for Southern states of India, as high yielding and rust resistant variety. It has all the characteristic features of JS 335 with an added advantage of resistance to rust and 10-12% yield superiority. It is the first of its kind in India developed and released by University of Agricultural Sciences, Dharwad. 
 

 

       
With this background work, the experiment on genetic analysis of rust resistance was conducted at the Main Agricultural Research Station (MARS), University of Agricultural Sciences, Dharwad during kharif 2016, summer 2017, kharif 2017 and kharif 2018. Resistant (EC 241778 and DSb 21) and susceptible genotypes (JS 335) were hybridised to generate F₁ plants during kharif 2016. The F₁’s of crosses were raised and true F₁ plants were identified based on phenotypic parental characteristics used in the crossing programme during summer 2017. The seeds from the identified true F₁ plants were used for raising F₂ plants in kharif 2017. Due care was taken for build-up of uniform and high inoculum pressure by planting one row of susceptible check after every five test rows of F₂ population and the plot was surrounded from all the sides by three rows of susceptible check. The observations on infection type for rust was recorded on parents, F₁ and F₂ plants on 0 to 9 scale as per standard evaluation system given by Mayee and Datar (1986). The severity of rust was scored between 75-90 days after sowing based on per cent leaf area infected by using 0-9 scale. This scale is based on lesion density, where 0: Immune (less than 1%), 1: highly resistant (1-10%), 3: moderately resistant (11-25%), moderately susceptible (26-50%), susceptible (50-75%) and highly susceptible (more than 75%). No fungicides were sprayed during the experiment.
       
The single seeds from the F₂ plants were used for raising F₃ progenies and were evaluated for rust resistance. The genotypes with score of ≤3 were considered as resistant. The ratio of resistant and susceptible reactions observed in the segregating population was tested for goodness-of-fit to theoretical ratios with the chi-square (χ²) test. 

Screening of germplasm
 
Among the germplasm lines screened under natural conditions only two lines viz., EC 241778 and EC 241780 consistently confirmed their resistance to rust during rainy seasons of 2002 and 2003. These lines showed highly resistant reaction (1 grade) with most of the top leaves free from rust pustules. These lines are in conformity with the findings of Patil and Basavaraja (1997). About 6 lines exhibited moderately resistant reaction (5 grade). The remaining lines showed susceptible (7 grade) to highly susceptible (9 grade) reaction.
 
Advanced breeding lines
 
The two exotic rust resistant lines and three popular varieties cultivated in Karanataka, which were highly susceptible to rust, viz., JS 335, JS 93-05 and DSb-1 were used in a long term breeding programme at Main Agricultural Research Station, University of Agricultural Sciences, Dharwad, Karnataka. The three promising lines thus developed viz., DSb 21, DSb 23-2 and DSB 28-3were further evaluated for their reaction to rust under natural epiphytotic condition from 2010 to 2013 (Table 2b) (Koraddi et al., 2017). Consistently in all the four years of screening these lines exhibited a score of 1 i.e., highly resistant reaction to rust, while the check, JS 335 exhibited a score of 9, indicating highly susceptible reaction to rust (Fig 2b, 3a and 3b).
 

 

 

 

 

       
Further these three promising lines were evaluated for their productivity under rust prone conditions at both the hotspots viz.,Ugarkhurd and Dharwad for four years i.e., from 2012 to 2013 along with popular variety, JS 335 as check. Under rust prone conditions, among these three lines evaluated, DSb 21 recorded highest seed yield of 1955 kg/ha, which is 157% increase over the check JS 335. Similarly, DSb 23-2 and DSb 28-3 recorded an average seed yield of 1918 kg/ha (152% increase) and 1768 kg/ha (132% increase) respectively (Table 3). 
 

       
These lines were also characterized for other traits like plant height, days to maturity, flower colour, pubescence and hilum colour. The results of which are presented in Table 4. All the three genotypes exhibited increased plant height compared to the check variety. Both DSb 21 and DSb 23-2 matured in 90-95 days while DSb 23-2 was late maturing which recorded a mean of 95-100 days. JS 335 was early maturing (85-90 days) compared to all the test entries. The three genotypes also exhibited variation in flower colour which could be used as one of the distinguishing morphological features of the three genotypes.
 

       
From among the three highly productive and rust resistant lines identified, DSb 21 entered into the All India Coordinated Trial during 2011 (Initial Varietal Trial). Later it was promoted to Advanced Varietal Trial I and II during 2012 and 2013 based on its superior performance and resistance to rust. The performance of DSb 21 in Southern Zone of All India Coordinated Trials (Karnataka, Tamil Nadu, Andhra Pradesh, Kerala and southern parts of Maharashtra Statesis presented in Table 5. From the table it is evident that DSb 21 recorded 17 per cent yield superiority over the check, JS 335. Based on the three years performance in coordinated trials it was released for Southern Zone of India during 44^th Annual Workshop of AICRP on soybean.
 

       
Apart from rust resistance and increased yield, the line DSb 21 exhibited no change in seed size (14.02 g/100 seeds) even under rust prone conditions compared to check JS 335, which exhibited drastic reduction in seed size (6.42 g/100 seeds) under rust prone conditions (Fig 3c). Further, DSb 21 recorded highest yield of 5250 kg/ha in one of the large scale demonstrations revealing its maximum genetic potentiality.
 

       
DSb 21 is the first of its kind in India developed by University of Agricultural Sciences, Dharwad. It has all the characteristic features of JS 335 (most popular cultivar) with an added advantage of resistance to rust and 10-12% yield superiority. Definitely, it will be a born to the soybean growing farmers by preventing significant yield losses due to soybean rust.
 
Inheritance studies
 
Information on the host differential response and genetics of resistance has lead to identification of six different rust resistance genes (Rpp: Resistance to P. pachyrhizi), named Rpp1 to Rpp6, against specific isolates of P. pachyrhizi (Hartman et al., 2005; Bonde et al., 2006; Miles et al., 2011). The reported six single dominant genes for specific resistance to P. pachyrhizi have been identified in different cultivars as Rpp1, Rpp2, Rpp3, Rpp4 (PI 459025) (Hartwig 1986), Rpp5 (Garcia et al., 2008) and Rpp6 (Shuxian et al., 2012). From these studies, it was clear that resistance to rust was pathogen race specific and also cultivar specific. To study the inheritance of rust resistance in newly released rust resistant variety, DSb 21, two crosses, JS 335 × EC 241778 (Susceptible × Resistant) and DSb 21 × JS 335 (Resistant × Susceptible) were developed. The two F₁ crosses, F₂ population and their F₃ progenies along with the resistant and susceptible parents were scored for rust resistance (Table 6a and Fig 4a). The results indicated that in both the crosses, the F₁’s exhibited resistance reaction with score less than or equal to 3. In both the F2 segregating populations, resistant and susceptible plants were recorded in the ratio of 3 (resistant):1 (susceptible), indicating resistance is dominant over susceptibility. The above results are in conformity with the findings of Bromfield and Hartwig (1980) who determined the inheritance of ASR resistance in two F₂ populations with PI 230970 and PI 230971 as the resistant parents. The analysis of these F₂’s showed that  rust resistance was dominant and qualitatively (simply) inherited. Other studies have reported partial to complete dominance action in the inheritance of rust resistance (Garcia et al., 2008; Ray et al., 2009). The result was also in line with the F₂ segregation analysis in six susceptible × resistant cross combinations which revealed that rust resistance is governed by a single dominant gene (Rahangdale and Raut 2004). Further to confirm the results of F₂ generation, plant to progeny rows of each F₂ plant from both the crosses were raised to generate F₃ progenies. The results of disease scoring in F₃ progenies indicated segregation of 1 (resistant) : 2 (segregating) : 1 (susceptible) type of reaction (Table 6b and Fig 4b), confirming the results of F₂ generation, that rust resistance in DSb 21 and EC 241778 are controlled by a single dominant gene. Similar results of inheritance of disease resistance controlled by single dominat gene were reported by Basamma et al., (2015) and Talukdar et al., (2013) in blackgram and soybean for mung bean yellow mosaic virus and yellow mosaic virus resistance respectively.
 

 

 

 

       
The identified new resistance source in this study will be a useful resource for efficiently breeding resistant soybean cultivars to soybean rust, improved yield and broadening the genetic base with stability of performance.

Rowing rust resistant soybean varieties is the most economical and efficient strategy currently adopted for management of soybean rust, even though chemical control has been recommended as the first line of defense. The fungus has several races with multiple virulence alleles which suggest that the ASR pathogen has high genetic variability. An attractive alternative to fungicide use to control the disease is the development of soybean cultivars with resistance to P. pachyrhizi. The genetic characterization of disease resistance in plants is essential for the understanding of plant pathogen interactions. These concepts contribute to an efficient breeding programme for disease resistance. DSb 21 is a highly productive and rust resistant soybean variety developed and released from University of Agricultural Sciences, Dharwad, Karnataka. The genetic analysis of disease resistance in this variety revealed that the resistance is controlled by a single dominant gene, which can be efficiently and easily used in the future breeding programme to develop new genotypes resistant to rust.

Author acknowledge the Department of Science and Technology for funding this research under Woman Scientist Scheme-A and Dr. G. T. Basavaraja, Principal Scientist (GPB), All India Coordinated Research Improvement Project on Soybean and MARS, Dharwad  for providing the material and necessary requirements for conducting the present study.