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Molecular and Phenotypic Assay of Improved Lines Harboring Bacterial Blight Resistance

1 Department of Plant Biotechnology, Center for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India 2 Center of Innovation, Dept. of Biotechnology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai, India 3 Department of Plant Breeding and Genetics, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai, India


Introduction
Bacterial blight is the most serious and destructive disease of many rice growing areas especially in south east Asia. It is caused by Xanthomonas oryzea pv. oryzae with the sign and symptoms of water-soaked lesions and wavy margins from tip of the leaf to the edge. It occurs in two phases; one is at seedling stage and another at maximum tillering stage.
The seedling stage symptoms are termed as 'kresek' and second phase symptoms starts as blightening of leaves at maximum tillering stage with distinct ooze out, in the form of

ISSN: 2319-7706 Volume 9 Number 8 (2020)
Journal homepage: http://www.ijcmas.com Bacterial blight (BB) is one of the serious and wide spread diseases of many rice cultivating areas in India as well as world. Development of cultivars pyramided with more than two BB resistance genes increases durable resistance to prevailing and emerging biotypes and it offers environment safe approach to manage the disease. The present study was carried out to screen the improved lines with the foreground markers of Xa21, xa13, xa5, Xa33 and Xa38 and the confirmed plants were evaluated for phenotypic resistance against the local isolate of BB under the controlled conditions. Further, the lines harboring BB resistance genes were evaluated for key-morphological traits to identify the superior genotypes. The bioassay revealed, most of the lines harboring Xa21, xa13, xa5andXa33/Xa38 in homozygous conditions were displayed high degree of resistance to bacterial blight with a mean lesion length (LL) ranged from 2.13 ± 0.41to 2.92 ± 0.65. The bioassay results also suggest, the lines harboring Xa21+ xa13 + xa5 + Xa33 resistance genes were observed to have less lesion length (2.13 ± 0.45) than Xa21+ xa13 +xa5 + Xa38 resistance genes (2.81 ± 0.40). Cluster analysis of 24 improved lines based on coefficient of genetic distance, identified the superior genotypes, which posses morphological characters as recurrent parents and also enriched with BB resistance genes. yellow droplets and it spread through water (Yasmin et al., 2017). The bacteria remain survive in the root of alternate host plant (Leesrsiaha xandra) and in infected paddy stubbles.
Rice is a staple crop for half of the world population with a production of 769.9 million tonnes and it is cultivated all over the world because of its wider adaptability to the vast environments. It is only the main source of carbohydrate for people below poverty line (Raveendra et al., 2020) and it is regarded as strategic crop under the food security. It has been affected by fungi, bacterial and viral diseases. Though, many diseases have been reported in rice, BB (Bacterial Blight) got its own significance, by infecting more than 50% of cultivated area.
During the severe outbreak of BB, it may cause a yield loss upto 60-75% (Kumar et al., 2012).For the control of BB, chemical method is effective only during the early stages, besides it also possesses the residual effect on soil. Host-plant resistance to BB is effective and it offers environment safe approach to control this disease (Ramalingam et al., 2017).
About 44BB resistance genes have been identified till date (Kim et al., 2018) and some of them were cloned. The resistance conferred by single gene can't be sufficient to defend the pathogen effectively, pyramiding of more than two effective resistance genes will gives broad-spectrum and durable resistance to BB than single gene resistance.
With this reference, the present study was carried out to evaluate the physical resistance of molecularly confirmed improved lines against bacterial blight disease under controlled conditions and also to assess the morphological traits to identify the phenotypically similar improved lines as recurrent parents.

Plant materials
Totally 24 lines of ImprovedBC 2 F 3 population for bacterial blight resistance (Xa21, xa13, xa5 and Xa33/Xa38) were developed in the backgrounds of ASD 16, ADT43 and ADT 47 at Tamil Nadu Agricultural University, Coimbatore. The present investigation was carried out at Agricultural College and Research Institute, Madurai to evaluate these improved lines for physical resistance against the BB under the controlled conditions as well as to evaluate the agro-morphological traits. The improved lines which possess these genes (Xa21, xa13, xa5and Xa33/Xa38) were confirmed by functional markers and were subjected to artificial bacterial blight resistant screening and same were assessed for agromorphological traits.

DNA Isolation
Fresh leaves from the improved lines and parent lines were collected from 6-8 weeks old seedlings and A was isolated by using CTAB method with slight modifications (Dellaporta, Wood, & Hicks, 1983). Quality of DNA was checked by using 0.8% agarose gel as well as quantified with spectrophotometer and final quantity of DNA was adjusted to 60ng/µl and stored at -20 0 C for further use.

PCR amplification
The improved lines which contain targeted genes (Xa21, xa13, xa5and Xa33/Xa38) were confirmed by using closely linked functional markers (Table 1). 10 µl of PCR mixture contain 1 µl of template DNA, 0.75 µl of dNTPs (2mM), 1 µl of 10x PCR buffer, 0.2 µl of Taq polymerase, 0.5 µl of each forward and reverse primers and 7 µl of sterile water. The PCR reaction was performed by using thermocycler (Eppendorf) with the following conditions: initial denaturation at 94 o c for 5 min followed by 35 cycles of final denaturation at 94 o c for 30sec; annealing at 56 o c for 30sec (Xa33) and 59 o c for 1min (Xa38); 72 o c for extention; 72 0 c for final extension. The amplified PCR products were separated by using 3% agarose gel stained with ethidium bromide and visualized through BIO-RADGel DOC XR+

Phenotypic screening for resistance against BB
From 24 lines of improved BC 2 F 3 population only 13 lines shown homozygous condition for all the targeted genes (Xa21, xa13 and xa5, Xa33/Xa38) were artificially inoculated with Xoo isolate. A total of three replications were maintained with 30 plants per each replication including parents as checks (IRBB60 as resistant; ASD 16, ADT 43 and ADT 47 as susceptible checks). To test the physical resistance of improved lines and parents against the BB, top five leaves were clipped off and leaves were dipped in to the suspension containing bacterial cells at a rate of 10 9 cells/ml by using clip inoculation method Kauffman, H. E. (1973). The symptoms were recorded 15 days after inoculation based on mean lesion length (LL) and scored according to the IRRI, SES IRRI, (2002). The plants with a LL (cm) of less than 5cm were considered as resistant and the plants with more than 5cm LL were recorded as Susceptible.

Morphological traits
The improved lines possessing the targeted genes (Xa21, xa13, xa5 andXa33/Xa38) were assessed for agronomical traits to check the performance of the pyramided lines over the parents. The data on morphological characters viz., days to 50% of flowering (DFF), plant height (PH), flag leaf length (FL), flag leaf width (FW),panicle length(PL), number of tillers (NT),number of productive tillers(NPT) and grain yield (SPY) were collected and statistically analyzed to identify the improved lines phenotypically similar to recurrent parents (ASD 16, ADT 43 and ADT 47).

Results and Discussion
The improved lines were screened for targeted genes (Xa21, xa13, xa5 and Xa33/Xa38) by using linked/functional markers (Figure 1; Table 1) and the positive plants were inoculated with the local isolate of BB to check for physical resistance in the controlled conditions. The bioassay of BB reveals, 13genotypically selected improved lines at BC 2 F 3 generation (Table 4) possessing Xa21, xa13, xa5 and Xa33/Xa38 in homozygous conditions exhibited a high level of resistance with a mean lesion length (cm) of 2.2 ± 0.89 to 2.9 ± 0.33with a diseases score of 0-1 (highly resistant). The rest of lines harboring Xa21, xa13 and xa5 without Xa33/Xa38 resistance genes, exhibited a mean lesion length ranged from 3.33± 0.41 to 3.92 ± 0.65 with a diseases score of 1-2.Similarly, the resistant check, IRBB60 (Xa21, xa13 andxa5) expressed a LL (cm) of 3.18 ± 0.44, while the susceptible checks ASD 16, ADT 43 and ADT 47 exhibited a mean lesion length of 15.12 ± 1.1, 15.57 ± 2.3 and 15.23 ± 1.3 respectively (Figure 4; Table 3). The evaluation of morphological traits revealed that, most of the improved lines were in similar with the recurrent parent morphological characters ( Table 2). Some of the lines viz., IL-16/60-3 in the background of ASD 16, IL-43/60-8-1 and IL-43/60-91 in the background of ADT 43 and IL-47/60-36 in the background of ADT 47 were produced higher yield than its respective recurrent parent. The cluster of selected improved lines suggests, all the improved lines (Table 2) in this study were in similar to their recurrent parents (Figure 2). Some promise lines viz., IL-16//60-3 in the background of ASD 16, IL-43/60-16-1 in the background of ADT 43 and IL-47/60-16-4 in the background of ADT 47 were highly similar to their recurrent parents ( Figure 2). Correlation of yield and yield contributing traits suggests, the number of productive tillers (r = 0.68) and number of tillers (r = 0.60) were correlated with grain yield (Figure 3). Development of cultivars with broad-spectrum resistance to BB highly essential to produce stable and higher yields. Developing cultivars with single gene is not recommended as it is overcome by newly evolved pathotypes.
To enhance the resistance levels to prevailing as well as emerging pathotypes, pyramiding of multiple genes in a single cultivar is highly essential for durable and broad-spectrum resistance (Pradhan et al., 2015;Ramalingam, Savitha, Ganesh, & Ranganathan, 2017;Sundaram et al., 2011). In the present investigation, physical resistance of selected improved lines possessing Xa21, xa13, xa5 and Xa33/Xa38 exhibited a mean lesion length (cm) of 2.81 ± 0.41 to 2.92 ± 0.65, while the resistant check, IRBB60 expressed 3.18 ± 0.44 lesion length (cm).In addition, the improved lines with Xa33/Xa38 resistance genes were displayed lesser lesion length than the lines with three resistance gene combination (Xa21, xa13 and xa5). While, in comparison of lines with Xa21 + xa13 + xa5 + Xa33 and Xa21 + xa13 + xa5 + Xa38, the Xa33 combination has given good results with lesser lesion length (2.13 ± 0.45 to 2.25 ± 0.25) than the lines with Xa38combination (2.81 ± 0.40 to 2.92 ± 0.65). Similar studies on NIL population in PB1121 for improved resistance against BB through introgression of Xa21+xa13 and Xa38 resistance genes and they evaluated for phenotypical screening against Xoo.
They have observed lesser lesion length of 3.83 ± 0.64, in the lines harboring Xa38 component in addition to Xa21 + xa13 (Ellur et al., 2016). Gidamo, 2015 has observed lesser lesion length in the lines harboring Xa33 resistance genes (0.4 ± 0.6) than Xa38 resistance genes (0.95 ± 0.69) phenotypical screening against BB (unpublished data).   3.18 ± 0.44 1 Lesion length (LL) of <5 cm is considered as resistant and lesion length of > 5 cm is considered susceptible.0-1 (Highly resistant); 9 (highly susceptible)  The results of Gidamo, 2015 supports the present study results, as the lines harboring Xa33 components was given lesser lesion length against isolates of Xoo. The resistance reaction of improved lines is due to the inheritance of resistant alleles from the donor parent, which were confirmed by using functional DNA markers (Figure 1). The morphological characters of the most of the improved lines were in similar with recurrent parent, due to the transfer of all the recurrent parent alleles for superior morphological characters as recurrent parents. The similar results were also obtained by (Hsu, Chiu, Yap, Tseng, & Wu, 2020), while introgressingXa4, xa5, Xa7, xa13 and Xa21 in to the TNG82 cultivar through markerassisted backcross breeding (MABB). Further, the improved lines with superior morphological characters and having physical resistance to BB were tested in multilocational trails to be release as new improved variety for BB prone rice cultivating areas or can be used as a potential donor in breeding programmes.