Kalyan K. Mondal

Improvement of Basmati rice varieties for resistance to blast and bacterial blight diseases using marker assisted backcross breeding.

Marker assisted backcross breeding was employed to incorporate the blast resistance genes, Pi2 and Pi54 and bacterial blight (BB) resistance genes xa13 and Xa21 into the genetic background of Pusa Basmati 1121 (PB1121) and Pusa Basmati 6. Foreground selection for target gene(s) was followed by arduous phenotypic and background selection which fast-tracked the recovery of recurrent parent genome (RPG) to an extent of 95.8% in one of the near-isogenic lines (NILs) namely, Pusa 1728-23-33-31-56, which also showed high degree of resemblance to recurrent parent, PB6 in phenotype. The phenotypic selection prior to background selection provided an additional opportunity for identifying the novel recombinants viz., Pusa 1884-9-12-14 and Pusa 1884-3-9-175, superior to parental lines in terms of early maturity, higher yield and improved quality parameters. There was no significant difference between the RPG recovery estimated based on SSR or SNP markers, however, the panel of SNPs markers was considered as the better choice for background selection as it provided better genome coverage and included SNPs in the genic regions. Multi-location evaluation of NILs depicted their stable and high mean performance in comparison to the respective recurrent parents. The Pi2+Pi54 carrying NILs were effective in combating a pan-India panel of Magnaporthe oryzae isolates with high level of field resistance in northern, eastern and southern parts of India. Alongside, the PB1121-NILs and PB6-NILs carrying BB resistance genes xa13+Xa21 were resistant against Xanthomonas oryzae pv. oryzae races of north-western, southern and eastern parts of the country. Three of NILs developed in this study, have been promoted to final stage of testing during the ​Kharif 2015 in the Indian National Basmati Trial.

Chitinase-mediated inhibitory activity of Brassica transgenic on growth of Alternaria brassicae.

Chitinase, capable of degrading the cell walls of invading phytopathogenic fungi, plays an important role in plant defense response, particularly when this enzyme is overexpressed through genetic engineering. In the present study, Brassica plant (Brassica juncea L.) was transformed with chitinase gene tagged with an overexpressing promoter 35 S CaMV. The putative transgenics were assayed for their inhibitory activity against Alternaria brassicae, the inducer of Alternaria leaf spot of Brassica both in vitro and under polyhouse conditions. In in vitro fungal growth inhibition assays, chitinase inhibited the fungal colony size by 12-56% over the non-trangenic control. The bioassay under artificial epiphytotic conditions revealed the delay in the onset of disease as well as reduced lesion number and size in 35S-chitinase Brassica as compared to the untransformed control plants.

Marker-aided Incorporation of Xa38 , a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21

Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India’s forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region.

The reliable and rapid polymerase chain reaction (PCR) diagnosis for Xanthomonas axonopodis pv. punicae in pomegranate

Bacterial blight is a major disease in pomegranate (Punica granatum) cultivation in India threatening the export potential of this important fruit crop. The disease is caused by a yellow pigmented, Gram negative, rod shaped bacterium, Xanthomonas axonopodis pv. punicae. We developed a polymerase chain reaction (PCR) based detection technique for this blight pathogen using primers designed from gyrB gene. A primer set KKM5 and KKM6 was synthesized based on sequence alignment of 530 nucleotides of C-terminus region in the gyrB genes from 15 different bacterial strains. The primer set was validated for amplification of 491 bp of gyrB gene. No amplification was observed in other phytopathogenic Xanthomonads including Xanthomonas axonopodis pv. citri, Xanthomonas axonopodis pv. phaseoli, Xanthomonas axonopodis pv. mangiferaeindicae, Xanthomonas campestris pv. manihotis, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Xanthomonas axonopodis pv. axonopodis and Pantoea agglomerans. The developed technique could detect the pathogen in infected pomegranate plant samples including leaf, fruit and stem within 3 h, at a detection limit 0.1 ng µl -1 template DNA.

Management of Xanthomonas camprestris pv. malvacearum-Induced Blight of Cotton Through Phenolics of Cotton Rhizobacterium

Four major phenolics were demonstrated to be produced by Pseudomonas fluorescens strain CRb-26, a cotton rhizobacterium antagonistic to Xanthomonas camprestris pv. malvacearum (Xcm), the inducer of bacterial blight of cotton. Of these, compounds II (nonfluorescent) and IV(fluorescent) completely inhibited the growth of Xcm in vitro. Among these, compound IV was produced maximally (39% of the four phenolics), and it protected cotton leaves from blight infection better than compound II under glass-house conditions. Compound IV, identified as 2,4-diacetylphloroglucinol, was, therefore, concluded to be a key metabolite involved in disease suppression by strain CRb-26 of P. fluorescens, which could be used as an ecofriendly potential input in the integrated management of bacterial blight of cotton.

Rice pathogen Xanthomonas oryzae pv. oryzae employs inducible hrp-dependent XopF type III effector protein for its growth, pathogenicity and for suppression of PTI response to induce blight disease

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice worldwide. Xoo secretes effector proteins (T3Es) directly into the rice cell through a hrp-encoded specialized type III secretion apparatus to induce blight. We examined the function of XopF, one of the conserved effectors in Xoo, using a null mutant developed through a PCR-based homologous recombination strategy. We studied inducible, hrp-dependent expression pattern of xopF. We confirmed that XopF is translocated in rice cytosol through T3SS using adenylate cyclase activity assay. XopF regulate the in planta Xoo growth and suppress PAMP-triggered immune (PTI) response in rice. Xoo wild but not mutant, Xoo ∆xopF produced intense blight lesions upon inoculation using leaf clipping method. Further, Xoo ∆xopF showed significant reduction in planta colonization relative to the wild strain. The relative expression analysis of PTI marker genes, PR10, OsWRKY13, OsRLCK16, and OsFLS2 indicated that these genes were up-regulated 1.5 to 5 fold upon challenge-inoculation with Xoo ∆xopF indicating the role of XopF in suppressing PTI in rice. Xoo ∆xopF mutant induced more callose deposition in infected rice leaves. XopF::EYFP fusion gene product was localized to the plasma membrane when transiently expressed in Nicotiana benthamiana as well as in living onion epidermal cells. Collectively, the present study shows that XopF repress basal PTI response in plants, and thus favours Xoo growth and pathogenicity in rice.

XopN-T3SS effector of Xanthomonas axonopodis pv. punicae localizes to the plasma membrane and modulates ROS accumulation events during blight pathogenesis in pomegranate

Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a major disease of pomegranate. Xap secretes effector proteins via type III secretion system (T3SS) to suppress pathogen-associated molecular pattern (PAMP)-triggered plant immunity (PTI). Previously we reported that XopN, a conserved effector of Xap, modulate in planta bacterial growth, and blight disease. In continuation to that here we report the deletion of XopN from Xap caused higher accumulation of reactive oxygen species (ROS) including H2O2 and O2-. We quantitatively assessed the higher accumulation of H2O2 in pomegranate leaves infiltrated with Xap ΔxopN compared to Xap wild-type. We analysed that 1.5 to 3.3 fold increase in transcript expression of ROS and flg22-inducible genes, namely FRK1, GST1, WRKY29, PR1, PR2 and PR5 in Arabidopsis when challenged with Xap ΔxopN; contrary, the up-regulation of all the genes were compromised when challenged with either Xap wild-type or Xap ΔxopN+xopN. Further, we demonstrated the plasma-membrane based localization of XopN protein both in its natural and experimental hosts. All together, the present study suggested that XopN-T3SS effector of Xap gets localized in the plasma membrane and suppresses ROS-mediated early defense responses during blight pathogenesis in pomegranate.

Genetic diversity of iturin producing strains of Bacillus species antagonistic to Ralstonia solanacerarum causing bacterial wilt disease in tomato

Bacillus spp. is a potential bacterial antagonist to manage bacterial wilt disease of tomato incited by Ralstonia solanacearum, which is one of the most threatening diseases of tomato in India. Genetic diversity of Bacillus strains and their potentiality to control bacterial wilt of tomato isolated from rhizospheric soil and endophytic tomato plants from different agro-climatic regions of India were studied. Rhizospheric soil and plants of tomato were pasteurized at 80°C for 15 min before dilution and then inoculated onto the Petri plates containing tryptic soy agar medium and incubated at 28± 2°C. Out of 250 isolates of Bacillus species, 47 strains showed antagonistic ability against R. solanacearum.

Xanthomonas axonopodis pv. punicae uses XopL effector to suppress pomegranate immunity

Xanthomonas axonopodis pv. punicae (Xap) causing bacterial blight is an important pathogen that incurs significant losses to the exportability of pomegranate. Xap uses the Xop TTSS-effector, via the type three secretion system, to suppress pomegranate immunity. Here, we investigate the role of XopL during blight pathogenesis. We observed that XopL is essential for its in planta growth and full virulence. Leaves inoculated with Xap ΔxopL produced restricted water-soaked lesions compared to those inoculated with wild-type Xap. XopL supports Xap for its sustained multiplication in pomegranate by suppressing the plant cell death (PCD) event. We further demonstrated that XopL suppresses immune responses, such as callose deposition and production of reactive oxygen species (ROS). RT-qPCR analysis revealed that immune responsive genes were upregulated when challenged with Xap ΔxopL, whereas upregulation of such genes was compromised in the complemented strain containing the xopL gene. The transiently expressed XopL::EYFP fusion protein was localized to the plasma membrane, indicating the possible site of its action. Altogether, this study highlights that XopL is an important TTSS-effector of Xap that suppresses plant immune responses, including PCD, presumably to support the multiplication of Xap for a sufficient time-period during blight disease development.

Development of Basmati rice genotypes with resistance to both bacterial blight and blast diseases using marker assisted restricted backcross breeding

Marker assisted backcross breeding (MABB) is aimed at introgression of trait(s) into a popular variety to augment specific trait(s) in an otherwise popular variety. While MABB can improve a variety with respect to introgressed trait(s), it offers very little scope for improvement of other traits. Marker assisted restricted backcross breeding (MARBB) is an alternative which can help in identifying transgressive segregants especially, when the donor parent is an elite genotype with several desirable traits. In the present study, restricted backcrossing followed by pedigree selection was used for the development of improved genotypes of Basmati rice with BB and blast diseases using an early maturing Basmati rice variety, Pusa Basmati 1509 as recurrent parent and an elite restorer line, Pusa 1790 as donor. Foreground selection for xa13, Xa21, Pi2 and Pi54 in the backcross progenies was combined with phenotypic selection for agronomic and grain quality traits to ensure premium Basmati grain quality in the progenies. Multilocation yield trial was conducted to evaluate the performance of the improved Basmati rice genotypes with both BB and blast resistance. Pusa 1847-12-62-115-20-6 and Pusa 1847-12-62-190-39-7 recorded significantly higher yields of 68.88 and 62.44 q/ha, respectively, compared to PB 1509 (57.88 q/ha). The improved progenies exhibited resistance to BB with an average lesion length of 2 cm, and blast with scores between 0–2, while PB 1509 was highly susceptible. Another genotype, Pusa 1847-12-62-37-8-3 exhibited head rice recovery (HRR) of 63.99%, which was significantly higher than in PB 1509 (56.40%). Marker assisted selection was also effected for fertility restoration genes and improved grain quality traits based on which two improved Basmati rice genotypes pyramided with BB and blast resistance namely, Pusa 1847-12-62-115-20-6 and Pusa 1847-12-62-190-39-7 were found promising, along with improved grain and cooking quality as well as restoration potential, which could be used in breeding better quality hybrids.