Molecular characterization and evaluation of novel management options for Burkholderia glumae BG1, the causative agent of panicle blight of rice (Oryza sativa L.).

BACKGROUND: Bacterial panicle blight, incited by Burkholderia glumae, has impacted rice production globally. Despite its significance, knowledge about the disease and the virulence pattern of the causal agent is very limited. Bacterial panicle blight is a major challenge in the rice-growing belts of North-western India, resulting in yield reduction. However, the management of B. glumae has become a challenge due to the lack of proper management strategies. METHODOLOGY AND RESULTS: Twenty-one BG strains have been characterized using the 16S rRNA and the gyrB gene-based sequence approach in the present study. The gyrB gene-based phylogenetic analysis resulted in geographic region-specific clustering of the BG isolates. The virulence screening of twenty-one BG strains by inoculating the pathogenic bacterial suspension of 1 × 10-8 cfu/ml at the booting stage (55 DAT) revealed the variation in the disease severity and the grain yield of rice plants. The most virulent BG1 strain resulted in the highest disease incidence (82.11%) and lowest grain yield (11.12 g/plant), and the least virulent BG10 strain resulted in lowest disease incidence of 18.94% and highest grain yield (24.62 g/plant). In vitro evaluation of various biocontrol agents and nano copper at different concentrations by agar well diffusion method revealed that nano copper at 1000 mg/L inhibited the colony growth of B. glumae. Under net house conditions, nano copper at 1000 mg/L reduced the disease severity to 21.23% and increased the grain yield by 20.91% (31.76 g per plant) compared to the positive control (COC 0.25% + streptomycin 200 ppm). Remarkably, pre-inoculation with nano copper at 1000 mg/L followed by challenge inoculation with B. glumae enhanced the activity of enzymatic antioxidants viz., Phenyl ammonia-lyase (PAL), Polyphenol oxidase (PPO) and Peroxidase (POX) and non-enzymatic antioxidant phenol. Additionally, we observed a substantial transcript level upregulation of six defense-related genes to several folds viz., OsPR2, OsPR5, OsWRKY71, OsPAL1, OsAPX1, and OsPPO1 in comparison to the pathogen control and healthy control. CONCLUSIONS: Overall, our study provides valuable insights into the potential and practical application of nano copper for the mitigation of bacterial panicle blight, offering promising prospects for commercial utilization in disease management.

Genetic Enhancement for Biotic Stress Resistance in Basmati Rice through Marker-Assisted Backcross Breeding.

Pusa Basmati 1509 (PB1509) is one of the major foreign-exchange-earning varieties of Basmati rice; it is semi-dwarf and early maturing with exceptional cooking quality and strong aroma. However, it is highly susceptible to various biotic stresses including bacterial blight and blast. Therefore, bacterial blight resistance genes, namely, xa13 + Xa21 and Xa38, and fungal blast resistance genes Pi9 + Pib and Pita were incorporated into the genetic background of recurrent parent (RP) PB1509 using donor parents, namely, Pusa Basmati 1718 (PB1718), Pusa 1927 (P1927), Pusa 1929 (P1929) and Tetep, respectively. Foreground selection was carried out with respective gene-linked markers, stringent phenotypic selection for recurrent parent phenotype, early generation background selection with Simple sequence repeat (SSR) markers, and background analysis at advanced generations with Rice Pan Genome Array comprising 80K SNPs. This has led to the development of Near isogenic lines (NILs), namely, Pusa 3037, Pusa 3054, Pusa 3060 and Pusa 3066 carrying genes xa13 + Xa21, Xa38, Pi9 + Pib and Pita with genomic similarity of 98.25%, 98.92%, 97.38% and 97.69%, respectively, as compared to the RP. Based on GGE-biplot analysis, Pusa 3037-1-44-3-164-20-249-2 carrying xa13 + Xa21, Pusa 3054-2-47-7-166-24-261-3 carrying Xa38, Pusa 3060-3-55-17-157-4-124-1 carrying Pi9 + Pib, and Pusa 3066-4-56-20-159-8-174-1 carrying Pita were identified to be relatively stable and better-performing individuals in the tested environments. Intercrossing between the best BC3F1s has led to the generation of Pusa 3122 (xa13 + Xa21 + Xa38), Pusa 3124 (Xa38 + Pi9 + Pib) and Pusa 3123 (Pi9 + Pib + Pita) with agronomy, grain and cooking quality parameters at par with PB1509. Cultivation of such improved varieties will help farmers reduce the cost of cultivation with decreased pesticide use and improve productivity with ensured safety to consumers.

Phylogenetic analysis, molecular characterization and virulence profiling based on toxoflavin gene of an Indian BG1 strain of Burkholderia glumae causing panicle blight of rice.

Bacterial panicle blight (BPB) caused by Burkholderia glumae (BG) has become significantly more prevalent in the rice-growing regions of North India. Based on virulence screening and in vitro quantification of toxoflavin, the BG strains were classified as hyper- (BG1 and BG3), moderate- (BG2, BG4, BG6, BG8, and BG9), and hypo- (BG5, BG7, and BG10) virulent. Plant inoculation assays with cell-free culture filtrate revealed strains with higher toxoflavin-producing ability had higher virulence. Based on 16S rRNA sequence, 6 isolates from Uttar Pradesh were grouped in clad C1; whereas, clad C2 exhibited 4 isolates, two each from Delhi and Uttar Pradesh. Strain BG1 being the most virulent Indian strain from Uttar Pradesh was further profiled for 11 tox genes. We found all the 11 tox genes present in strain BG1. In toxRABCDE cluster, all tox genes showed high similarity to B. glumae BGR1 except toxB, whereas in toxFGHIJ cluster toxF, toxG, toxH and toxI shared maximum similarity to B. glumae 336gr-1. tox genes of BG1 exhibited homology as well as divergence with B. gladioli. The domain prediction and protein association network analysis indicated the possible involvement of tox genes in the toxoflavin biosynthesis. As per our knowledge, this is the first report in India on characterization of tox genes cluster in B. glumae. Altogether, our study unravels a reliable method for identifying and characterizing B. glumae using tox genes and its relationship with disease production. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03660-6.

Complete genome resource unravels the close relation of an Indian Xanthomonas oryzae pv. oryzae strain IXOBB0003 with Philippines strain causing bacterial blight of rice.

Xanthomonas oryzae pv. oryzae (Xoo) is a pathogen of concern for rice growers as it limits the production potential of rice varieties worldwide. Due to their high genomic plasticity, the pathogen continues to evolve, nullifying the deployed resistance mechanisms. It is pertinent to monitor the evolving Xoo population for the virulent novel stains, and the affordable sequencing technologies made the task feasible with an in-depth understanding of their pathogenesis arsenals. We present the complete genome of a highly virulent Indian Xoo strain IXOBB0003, predominantly found in northwestern parts of India, by employing next-generation sequencing and single-molecule sequencing in real-time technologies. The final genome assembly comprises 4,962,427 bp and has 63.96% GC content. The pan genome analysis reveals that strain IXOBB0003 houses total of 3655 core genes, 1276 accessory genes and 595 unique genes. Comparative analysis of the predicted gene clusters of coding sequences and protein count of strain IXOBB0003 depicts 3687 of almost 90% gene clusters shared by other Asian strains, 17 unique to IXOBB0003 and 139 CDSs of IXOBB0003 are shared with PXO99A. AnnoTALE-based studies revealed 16 TALEs conferred from the whole genome sequence. Prominent TALEs of our strain are found orthologous to TALEs of the Philippines strain PXO99A. The genomic features of Indian Xoo strain IXOBB0003 and in comparison with other Asian strains would certainly contribute significantly while formulating novel strategies for BB management. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03596-x.

Amelioration of cold-induced sweetening in potato by RNAi mediated silencing of StUGPase encoding UDP-glucose pyrophosphorylase.

Cold-induced sweetening (CIS) is an unwanted physiological phenomenon in which reducing sugars (RS) get accumulated in potato (Solanum tuberosum) upon cold storage. High RS content makes potato commercially unsuitable for processing due to the unacceptable brown color in processed products like chips, fries, etc., and the production of a potential carcinogen, acrylamide. UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose towards the synthesis of sucrose and is also involved in the regulation of CIS in potato. The objective of the present work was RNAi-mediated downregulation of the StUGPase expression level in potato for the development of CIS tolerant potato. Hairpin RNA (hpRNA) gene construct was developed by placing UGPase cDNA fragment in sense and antisense orientation intervened by GBSS intron. Internodal stem explants (cv. Kufri Chipsona-4) were transformed with hpRNA gene construct, and 22 transgenic lines were obtained by PCR screening of putative transformants. Four transgenic lines showed the highest level of RS content reduction following 30 days of cold storage, with reductions in sucrose and RS (glucose & fructose) levels of up to 46% and 57.5%, respectively. Cold stored transgenic potato of these four lines produced acceptable chip colour upon processing. The selected transgenic lines carried two to five copies of the transgene. Northern hybridization revealed an accumulation of siRNA with a concomitant decrease in the StUGPase transcript level in these selected transgenic lines. The present work demonstrates the efficacy of StUGPase silencing in controlling CIS in potato, and the strategy can be employed for the development of CIS tolerant potato varieties.

Rice transcriptome upon infection with Xanthomonas oryzae pv. oryzae relative to its avirulent T3SS-defective strain exposed modulation of many stress responsive genes.

Xanthomonas oryzae pv. oryzae (Xoo) is a destructive pathogen that causes bacterial blight disease of rice worldwide. Xoo uses T3SS (type III secretion system) effectors to subvert rice innate immunity. However, the comprehensive knowledge of rice genes involved in T3SS effectors-mediated interaction remains unclear. In this study, the transcriptome profiles of rice infected with a virulent Xoo strain from North-eastern region of India relatives to its avirulent strain (that lacks functional T3SS) were analyzed at early (2-6 hpi) and late (16-24 hpi) hours of infection. Out of total 255 differentially expressed genes (DEGs), during early infection, 62 and 70 genes were upregulated and downregulated, respectively. At late infection, 70 and 53 genes were upregulated and downregulated, respectively. The transcriptomic data identified many differentially expressed resistant genes, transposons, transcription factors, serine/threonine protein kinase, cytochrome P450 and peroxidase genes that are involved in plant defense. Pathway analysis revealed that these DEGs are involved in hormone signaling, plant defense, cellular metabolism, growth and development processes. DEGs associated with plant defense were also validated through quantitative real-time PCR. Our study brings a comprehensive picture of the rice genes that are being differentially expressed during bacterial blight infection. Nevertheless, the DEG-associated pathways would provide sensible targets for developing resistance to bacterial blight. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03193-4.

Xanthomonas axonopodis pv. punicae depends on multiple non-TAL (Xop) T3SS effectors for its coveted growth inside the pomegranate plant through repressing the immune responses during bacterial blight development.

Xanthomonas axonopodis pv. punicae (Xap), the bacterial blight pathogen of pomegranate, incurs substantial loss to yield and reduces export quality of this economically important fruit crop. During infection, the bacterium secretes six non-TAL (Xop) effectors into the pomegranate cells through a specialized type three secretion system (T3SS). Previously, we demonstrated the role of two key effectors, XopL and XopN in pathogenesis. Here, we investigate the role of rest effectors (XopC2, XopE1, XopQ and XopZ) on disease development. We generated null mutants for each individual effector and mutant bacterial suspension was infiltrated into pomegranate leaves. Compared to Xap wild, the mutant bacterial growth was reduced by 2.7-11.5 folds. The mutants produced lesser water-soaked lesions when infiltrated on leaves by 1.13-2.21 folds. Among the four effectors, XopC2 contributes highest for in planta bacterial growth and disease development. XopC2 efficiently suppressed the defense responses like callose deposition, reactive oxygen species (ROS) and the activation of immune responsive genes. Being a major contributor, we further characterize XopC2 for its subcellular localization, its protein structure and networking. XopC2 is localized to the plasma membrane of Nicotiana benthamiana like XopL and XopN. XopC2 is a 661 amino acids protein having 15 alpha and 17 beta helix. Our STRING and I-TASSER based analysis hinted that XopC2 interacts with multiple membrane localized plant proteins including transcription regulator of CCR4-NOT family, TTN of maintenance of chromosome family and serine/threonine-protein phosphatase 2A (PP2A) isoform. Based on the interaction it is predicted that XopC2 might involve in diverse functions like nuclear-transcribed mRNA catabolic process, maintenance of chromosome, hormone signaling and protein dephosphorylation activities and thereby suppress the plant immunity. Altogether, our study suggests that Xap largely depends on three non-TAL (Xop) effectors, including XopC2, XopL and XopN, to modulate pomegranate PTI for its unrestricted proliferation during bacterial blight development.

Complete Genome Sequence of Indian Race 4 of Xanthomonas oryzae pv. oryzae, the Causal Agent of Bacterial Blight of Rice.

Xanthomonas oryzae pv. oryzae, the causal bacterium of bacterial blight limits rice production globally. Currently, genome sequences for only a few X. oryzae pv. oryzae isolates are available from India. Based on the next-generation sequencing and single-molecule sequencing in real-time technologies, we present here the complete genome sequence of X. oryzae pv. oryzae race 4, a highly virulent member of the Indian X. oryzae pv. oryzae population that has been extensively used in different research studies. The genome data will contribute to our understanding of X. oryzae pv. oryzae genomic features and pave the way for research on rice-X. oryzae pv. oryzae interactions.

XopR T3SS-effector of Xanthomonas oryzae pv. oryzae suppresses cell death-mediated plant defense response during bacterial blight development in rice.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease that limits the rice production globally. The bacterium secretes effector proteins directly into plant cells through a type III secretion system (T3SS). Here, we examined the role of a conserved XopR T3SS-effector in the suppression of host basal defense response. Phylogenetic and sequence analysis showed that XopR is well conserved within Xoo strains but shares varying degree of similarity among the other Xanthomonas species. The expression of XopR was shown to be regulated by hrpX, a key regulator of hrp cluster. For functional analysis we employed two mutant strains of Xoo, one lacks xopR gene and other lacks hrpX gene (making the strain defective in T3SS). Programmed cell death (PCD) events was examined both in rice and tobacco leaves through trypan blue staining method. In XopR expressing tobacco leaves the PCD induction was compromised. We observed higher PCD on rice leaves inoculated with Xoo mutants lacking either xopR or functional T3SS as compared to wild type. Contrary, when xopR gene was complemented in mutated strain the PCD was suppressed which clearly suggests that XopR acts as suppressor of the PCD mediated defense response. The EYFP::XopR fusion protein was shown to be localized to the plasma membrane of Nicotiana benthamiana and onion epidermal cells. Altogether our study leads to the understanding that XopR T3SS-effector is essential for Xoo to suppress PCD, primarily to support the in planta colonization of Xoo during blight pathogenesis.

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.

XopR TTSS-effector regulates in planta growth, virulence of Indian strain of Xanthomonas oryzae pv. oryzae via suppressing reactive oxygen species production and cell wall-associated rice immune responses during blight induction.

Xanthomonas oryzae pv. oryzae (Xoo) causing bacterial blight of rice is a global problem in rice production. Phytopathogenic Xanthomonads overpower PAMP-triggered immunity (PTI) through secreting effectors via type III secretion system (TTSS). We previously screened the TTSS effector repository of an Indian strain of Xoo (race 4), a predominant strain from north-west India that contains 21 Xop and 18 TALE effectors. Here, we demonstrate that Xoo race 4 employs XopR for in planta colonisation, virulence and for the suppression of cell wall-associated immune responses in its natural host. XopR null mutant (Xoo ΔxopR) produced 2.6-fold less-severe lesion as compared with Xoo wild type. Xoo ΔxopR showed 1.58-fold reduced colonisation compared with wild indicating that XopR is required for maximum colonisation in rice. Xoo ΔxopR produced 3.8-fold more callose deposits compared with wild. Xoo ΔxopR caused significantly higher production of ROS in rice. RT-qPCR expression analysis of immune responsive genes of rice indicated 10- to 43-fold upregulation upon challenged inoculation with Xoo ΔxopR over wild. Altogether, our study revealed that XopR of Indian Xoo strain supports its in planta growth and contributes immensely for successful blight development through suppressing defence related events like reactive oxygen species production, callose deposition and transcript abundance of immune responsive genes during rice::Xoo interaction.

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.

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.

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.

General polynomial factorization-based design of sparse periodic linear arrays.

We have developed several methods of designing sparse periodic arrays based upon the polynomial factorization method. In these methods, transmit and receive aperture polynomials are selected such that their product results in a polynomial representing the desired combined transmit/receive (T/R) effective aperture function. A desired combined T/R effective aperture is simply an aperture with an appropriate width exhibiting a spectrum that corresponds to the desired two-way radiation pattern. At least one of the two aperture functions that constitute the combined T/R effective aperture function will be a sparse polynomial. A measure of sparsity of the designed array is defined in terms of the element reduction factor. We show that elements of a linear array can be reduced with varying degrees of beam mainlobe width to sidelobe reduction properties.

ERIC-PCR-generated genomic fingerprints and their relationship with pathogenic variability of Xanthomonas campestris pv. punicae, the incitant of bacterial blight of pomegranate.

Bacterial blight caused by Xanthomonas campestris pv. punicae (Xcp) has emerged as a potential threat in pomegranate (Punica granatum) cultivation in India. Here, we report the genomic fingerprints and their correlation with virulence pattern of Xcp isolates from Maharashtra and Delhi. The genomic fingerprints of Xcp isolates were generated using enterobacterial repetitive intergenic consensus (ERIC) sequence-based primers, and virulence level was based on their reaction upon infiltration to susceptible pomegranate cultivar. Maharashtra isolate PGM1 showed only 50% similarity with Delhi isolate PGD8 forming a distinct genotype, whereas the Delhi isolates PGD5 and PGD6 form a cluster with Maharashtra isolates PGM2 and PGM4. The isolates PGM2, PGM4, PGD5, and PGD6 showing mean disease score of 7.47 were marked as group A or highly virulent. The moderately virulent or group B isolates PGM3 and PGD7 produced mean disease score of 4.19, whereas less virulent or group C isolates PGD8 and PGM1 gave mean disease intensity of 1.91. A correlation between genotypic groups based on ERIC fingerprints and pathogenicity of the isolates was established. The highly virulent isolates PGM2, PGM4, PGD5, and PGD6 formed a single cluster. A unique 900 bp amplicon present in all highly virulent isolates has been identified that can be used as genetic marker to screen isolates for virulence. The less virulent isolates PGD8 and PGM1 formed single cluster at 50% similarity coefficient. This seems to be the first report to establish a correlation between ERIC-PCR fingerprints and their corresponding virulence pattern of the pomegranate bacterial blight pathogen.

Transgenic Indian mustard (Brassica juncea) expressing tomato glucanase leads to arrested growth of Alternaria brassicae.

Brassica juncea is an important oilseed crop of the Indian sub-continent. Yield loss due to fungal disease alternaria leaf spot caused by Alternaria brassicae is a serious problem in cultivation of this crop. Nonavailability of resistance genes within crossable germplasms of Brassica necessitates use of genetic engineering strategies to develop genetic resistance against this pathogen. The pathogenesis related (PR) proteins are group of plant proteins that are toxic to invading fungal pathogens, but are present in plant in trace amount. Thus, overexpression of PR proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein glucanase hydrolyzes a major cell-wall component, glucan, of pathogenic fungi and acts as a plant defense barrier. We report the expression of a class I basic glucanase gene, under the control of CaMV 35S promoter, in Indian mustard and its genetic resistance against alternaria leaf spot. Southern and Northern hybridization confirmed stable integration and expression of the glucanase gene in mustard transgenics. Several independent transgenics were screened in vitro and under poly house conditions for their resistance against Alternaria brassicae. In an in vitro antifungal assay, transgenics arrested hyphal growth of Alternaria brassicae by 15-54%. Under pathogen-challenged conditions in poly house, the transgenics showed restricted number, size and spread of lesions caused by Alternaria brassicae. Also, the onset of disease was delayed in transgenics compared to untransformed parent plants. The results demonstrate potentiality of a PR protein from a heterologous source in developing alternaria leaf spot resistance in Indian mustard.

Use of a chiral surfactant for enantioselective reduction of a ketone.

The influence of a chiral surfactant and a polymer-supported chiral additive on reduction of ketones using sodium borohydride will be described. Initial preparations involved methylation of (S)-leucinol to give (2S)-N , N-dimethyl-2-amino-4-methyl-1-pentanol (1) (67%). The chiral surfactant (2) was synthesized by reacting (1) with bromohexadecane (71%). The functionalized styrene for the polymer-supported chiral additive (5) was synthesized by reacting (1) with 4-vinylbenzyl chloride. Polymerization was carried out with 10% of the functionalized monomer (4), 5% cross-linking agent divinylbenzene, and 85% styrene with AIBN as the initiator. The activity of the chiral surfactant and polymeric additive were examined by using them as additives in a standard reduction of 2-pentanone with sodium borohydride to yield (R)- and (S)-2-pentanol (3) (20%). The resulting alcohol was analyzed by polarimetry (ee 9.5%) and also esterified with (2S)-methylbutyric acid prior to characterization by NMR. 13C NMR indicated an enantiomeric excess of 5.2% when the chiral surfactant was used, and 7% when the polymeric additive was used.

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.

Propagation of dust-acoustic waves in a bounded dusty plasma.

The linear and nonlinear propagation of dust-acoustic waves in a dusty plasma bounded in finite geometry has been theoretically investigated. It is found that the finite geometry of the bounded plasma makes a significant contribution to the instability of the wave. Moreover, the pseudopotential has a positive and inverted profile that prevents the trapping of particles and does not favor solitary waves in bounded dusty plasma.