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 diversity and virulence of phytopathogenic Burkholderia glumae strains isolated from rice cultivars in valleys of the high jungle of Peru.

Burkholderia glumae causes bacterial leaf blight in rice, and its global spread has been exacerbated by climate change. To understand the genetic diversity and virulence of B. glumae strains isolated from rice cultivars in Peru, 47 isolates were obtained from infected rice fields, all belonging to B. glumae, and confirmed by recA and toxB sequences. The BOX-PCR typing group 38 genomic profiles, and these turn into 7 Variable Number Tandem Repeats (VNTR) haplotypes. There was no correlation between clustering and geographical origin. Nineteen strains were selected for phenotypic characterization and virulence, using both the maceration level of the onion bulb proxy and inoculation of seeds of two rice cultivars. Several strains produced pigments other than toxoflavin, which correlated with onion bulb maceration. In terms of virulence at the seed level, all strains produced inhibition at the root and coleoptile level, but the severity of symptoms varied significantly between strains, revealing significant differences in pathogenicity. There is no correlation between maceration and virulence scores, probably reflecting different virulence mechanisms depending on the host infection stage. This is the first study to evaluate the VNTR diversity and virulence of Peruvian strains of B. glumae in two commercial cultivars.

A novel salivary effector, BtE3, is essential for whitefly performance on host plants.

The whitefly Bemisia tabaci is a piercing-sucking herbivore that reduces the yields of crops both by feeding on plants and transmitting plant viruses. Like most plant feeders, B. tabaci has evolved ways to avoid plant defence responses. For example, B. tabaci is known to secrete salivary effectors to suppress host defences. However, the nature of B. tabaci effectors is not completely understood. In this study, we used B. tabaci genomic and salivary gland transcriptomic data and an overexpression system to identify a previously unknown B. tabaci salivary effector, BtE3. BtE3 is specifically expressed in the head (containing primary salivary glands) and is secreted into hosts during B. tabaci feeding. In planta overexpression of BtE3 blocked Burkholderia glumae-induced hypersensitive response (HR) in both Nicotiana benthamiana and Solanum lycopersicum. Silencing of BtE3 by plant-mediated RNAi prevented B. tabaci from continuously ingesting phloem sap, and reduced B. tabaci survival and fecundity. Moreover, overexpression of BtE3 in planta up-regulated the salicylic acid- (SA-) signalling pathway, but suppressed the downstream jasmonic acid- (JA-) mediated defences. Taken together, these results indicate that BtE3 is a B. tabaci-specific novel effector involved in B. tabaci-plant interactions. These findings increase our understanding of B. tabaci effectors and suggest novel strategies for B. tabaci pest management.

N-acylhomoserine lactonase-based hybrid nanoflowers: a novel and practical strategy to control plant bacterial diseases.

BACKGROUND: The disease caused by plant pathogenic bacteria in the production, transportation, and storage of many crops has brought huge losses to agricultural production. N-acylhomoserine lactonases (AHLases) can quench quorum-sensing (QS) by hydrolyzing acylhomoserine lactones (AHLs), which makes them the promising candidates for controlling infections of QS-dependent pathogenic bacteria. Although many AHLases have been isolated and considered as a potentially effective preventive and therapeutic agents for bacterial diseases, the intrinsically poor ambient stability has seriously restricted its application. RESULTS: Herein, we showed that a spheroid enzyme-based hybrid nanoflower (EHNF), AhlX@Ni3(PO4)2, can be easily synthesized, and it exhibited 10 times AHL (3OC8-HSL) degradation activity than that with free AhlX (a thermostable AHL lactonase). In addition, it showed intriguing stability even at the working concentration, and retained ~ 100% activity after incubation at room temperature (25 °C) for 40 days and approximately 80% activity after incubation at 60 °C for 48 h. Furthermore, it exhibited better organic solvent tolerance and long-term stability in a complicated ecological environment than that of AhlX. To reduce the cost and streamline production processes, CSA@Ni3(PO4)2, which was assembled from the crude supernatants of AhlX and Ni3(PO4)2, was synthesized. Both AhlX@Ni3(PO4)2 and CSA@Ni3(PO4)2 efficiently attenuated pathogenic bacterial infection. CONCLUSIONS: In this study, we have developed N-acylhomoserine lactonase-based hybrid nanoflowers as a novel and efficient biocontrol reagent with significant control effect, outstanding environmental adaptability and tolerance. It was expected to overcome the bottlenecks of poor stability and limited environmental tolerance that have existed for over two decades and pioneered the practical application of EHNFs in the field of biological control.

Complete Genome Sequence Data of Four Burkholderia glumae Strains Isolated from Rice Fields in the United States.

Bacterial panicle blight caused by Burkholderia glumae is a major disease in rice production worldwide. Currently, only a few whole-genome sequences of B. glumae strains isolated in the United States are available. Here, we report the complete genome sequence of four B. glumae strains, including three virulent strains (336gr-1, 411gr-6, and 957856-41-c) and the nonpathogenic strain B. glumae 257sh-1, which were isolated from rice fields in Louisiana (336gr-1, 957856-41-c, and 257sh-1) and Arkansas (411gr-6). The whole-genome sequence data of B. glumae strains will contribute to investigations of the molecular mechanism underlying bacterial pathogenicity and virulence to rice plants.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A system to study the expression of phytopathogenic genes encoded by Burkholderia glumae.

Rice is often infected by bacterial panicle blight disease caused by Burkholderia glumae. Since most studies have assessed the transcriptome of the plant when it is exposed to bacteria, the gene expression of the phytopathogenic bacteria have not been well elaborated during the infection process or in the host cell. Recently, a few researches were conducted to evaluate the in vivo transcriptome of bacteria during the infective process. Most bacterial cells do not express genes involved in pathogenicity in culture medium making it difficult to investigate gene expression of bacterial cells in plant cells. Here, we sought a simulated patho-system that would allow bacterial cells to express their pathogenic genes. Thus, rice root exudates (RE) and bacterial N-acyl homoserine lactone (AHL) were used and their effects on bacterial gene expression were assessed. Transcription patterns of B. glumae virulence determinants showed that enrichment medium (LB + RE + C8-HSL) could significantly induce virulence factor genes compared with Luria Bertani (LB; control) medium. The data indicate that the artificial environment is similar to the real patho-system, and that this induced maximum relevant gene expression. In this model system, bacterial gene expression changes are traceable in the infection process. Bacterial cells exposed to either an artificial environment or LB + RE + C8-HSL behaved similarly to the natural environment in situ.

Isolation and Characterization of Bacteriophages Infecting Burkholderia glumae, the Major Causal Agent of Bacterial Panicle Blight in Rice.

Bacterial panicle blight (BPB), caused by Burkholderia glumae, is one of the most severe seed-borne bacterial diseases of rice in the world, which can decrease rice production by ≤75%. Nevertheless, there are few effective measures to manage this disease. In an attempt to develop an alternative management tool for BPB, we isolated and characterized phages from soil and water that are effective to lyse several strains of B. glumae. After tests of host ranges, the phages NBP1-1, NBP4-7, and NBP4-8 were selected for further comprehensive characterization, all of which could lyse B. glumae BGLa14-8 (phage sensitive) but not B. glumae 336gr-1 (phage insensitive). This result indicates that the phages killing B. glumae cells have specific host ranges at the strain level within the bacterial species. In the greenhouse condition of this study, foliar application of the phage NBP4-7 reduced the severity of BPB caused by B. glumae BGLa14-8 ≤62% but did not cause any significant effect on the infection by B. glumae 336gr-1. Electron microscopy and whole-genome sequencing were also performed to characterize the three selected phages. Transmission electron microscopy revealed that the selected phages belong to the family Myoviridae. Furthermore, whole-genome sequence analysis indicated that the three phages belong to a same species and are closely related to the Burkholderia phage KL3, a member of the Myoviridae family.

VNTR Typing of the Bacterial Rice Pathogen Burkholderia glumae Reveals the Coexistence of Several Diverging Lineages in a Single Field in Colombia.

Burkholderia glumae is responsible for the panicle blight disease of rice. This disease is present worldwide and can result in significant drop in yields. To estimate the genetic diversity of the bacterial strains present in a rice paddy field in Colombia, we sampled 109 strains from infected panicles. To detect fine genetic relationships among related haplotypes, and to overcome a very low nucleotide diversity detected in previous studies, we designed primers to amplify and sequence several highly variable minisatellite loci, or variable number tandem repeats (VNTRs), as well as part of the Toxoflavin toxA gene in all strains. Results show that the toxA nucleotide diversity defined four lineages and was similar to that detected in several fields in Japan; data suggest that B. glumae has spread from Asia to America without major loss of genetic diversity, and that five VNTR loci discriminated the strains within the field revealing single and multi-infections of the rice panicles with a wide distribution of the haplotypes among the different plots. Even though disease levels vary considerably from year to year, the bacterial genetic diversity is maintained within a field. We do not detect any geographical structuring within the field, nor any effect of the rice cultivar on the observed diversity. The consequences on the origin and evolution of the bacteria are discussed.

The In Vitro and In Planta Interspecies Interactions Among Rice-Pathogenic Burkholderia Species.

Burkholderia glumae, B. plantarii, and B. gladioli are responsible for serious diseases in rice crops and co-occurrence among them has been reported. In this study, in vitro assays revealed antagonistic activity among these organisms, with B. gladioli demonstrating strong inhibition of B. glumae and B. plantarii. Strains of B. glumae and B. plantarii that express green fluorescent protein were constructed and used for cocultivation assays with B. gladioli, which confirmed the strong inhibitory activity of B. gladioli. Cell-free supernatants from each species were tested against cultures of counterpart species to evaluate the potential to inhibit bacterial growth. To investigate the inhibitory activity of B. gladioli on B. glumae and B. plantarii in rice, rice plant assays were performed and quantitative PCR (qPCR) assays were developed for in planta bacterial quantification. The results indicated that coinoculation with B. gladioli leads to significantly reduced disease severity and colonization of rice tissues compared with single inoculation with B. glumae or B. plantarii. This study demonstrates the interactions among three rice-pathogenic Burkholderia species and strong antagonistic activity of B. gladioli in vitro and in planta. The qPCR assays developed here could be applied for accurate quantification of these organisms from in planta samples in future studies.

Genetic Diversity and Distribution of Korean Isolates of Burkholderia glumae.

Burkholderia glumae causes panicle blight of rice (grain rot in Japan and Korea), and the severity of damage is increasing worldwide. During 2017 and 2018, 137 isolates of B. glumae were isolated from symptomatic grain rot of rice cultivated in paddy fields throughout South Korea. Genetic diversity of the isolates was determined using transposase-based PCR (Tnp-PCR) genomic fingerprinting. All 138 isolates, including the B. glumae BGR1 strain, produced toxoflavin in various amounts, and 17 isolates produced an unidentified purple or orange pigment on Luria-Bertani medium and casamino acid-peptone-glucose medium, respectively, at 28°C. Transposase-based PCR genomic fingerprinting was performed using a novel primer designed based on transposase (tnp) gene sequences located at the ends of the toxoflavin efflux transporter operon; this method provided reliable and reproducible results. Through Tnp-PCR genomic fingerprinting, the genetic groups of Korean B. glumae isolates were divided into 11 clusters and three divisions. The Korean B. glumae isolates were mainly grouped in division I (73%). Interestingly, most of the pigment-producing isolates were grouped in divisions II and III; of these, 10 were grouped in cluster VIII, which comprised 67% of this cluster. Results of a phylogenetic analysis based on tofI and hrpB gene sequences were consistent with classification by Tnp-PCR genomic fingerprinting. The BGR1 strain did not belong to any of the clusters, indicating that this strain does not exhibit the typical genetic representation of B. glumae. B. glumae isolates showed diversity in the use of carbon and nitrogen sources, but no correlation with genetic classification by PCR fingerprinting was found. This is the first study to analyze the geographical distribution and genetic diversity of Korean B. glumae isolates.

Harnessing Pseudomonas protegens to Control Bacterial Panicle Blight of Rice.

Bacterial panicle blight of rice is a seedborne disease caused by the bacterium Burkholderia glumae. This disease has affected rice production worldwide and its effects are likely to become more devastating with the continuous increase in global temperatures, especially during the growing season. The bacterium can cause disease symptoms in different tissues and at different developmental stages. In reproductive stages, the bacterium interferes with grain development in the panicles and, as a result, directly affects rice yield. Currently, there are no methods to control the disease because chemical control is not effective and completely resistant cultivars are not available. Thus, a promising approach is the use of antagonistic microorganisms. In this work, we identified one strain of Pseudomonas protegens and one strain of B. cepacia with antimicrobial activity against B. glumae in vitro and in planta. We further characterized the antimicrobial activity of P. protegens and found that this activity is associated with bacterial secretions. Cell-free secretions from P. protegens inhibited the growth of B. glumae in vitro and also prevented B. glumae from causing disease in rice. Although the specific molecules associated with these activities have not been identified, these findings suggest that the secreted fractions from P. protegens could be harnessed as biopesticides to control bacterial panicle blight of rice.

Evaluation of major rice cultivars for resistance to bacterial seedling rot caused by Burkholderia glumae and identification of Japanese standard cultivars for resistance assessments.

Burkholderia glumae causes bacterial seedling rot (BSR) and bacterial grain rot (BGR) in rice (Oryza sativa), both of which are important diseases in Japan. We previously evaluated major Japanese cultivars for BGR resistance and selected standard cultivars for resistance assessments. Here, we assessed the BSR occurrence rate in cultivars from the World Rice Collection (WRC) and other sources and found wide variation in resistance. Next, we evaluated major Japanese cultivars for BSR resistance and found that two Japanese landraces, 'Kujuu' and 'Aikoku', showed "strong" resistance; most others were categorized as "medium" or "medium to weak". We previously developed a near-isogenic line (RBG1-NIL) by introducing the genomic region containing RBG1, a quantitative trait locus (QTL) for BSR resistance, from 'Nona Bokra' (indica) into 'Koshihikari' (temperate japonica). The resistance level of RBG1-NIL was "strong", indicating the effectiveness of RBG1 against BSR. The correlation between BSR and BGR resistance scores was low, indicating that it is necessary to introduce QTLs for resistance from different sources to develop cultivars resistant to both BSR and BGR. On the basis of the screening results, we selected standard cultivars for BSR resistance to cover a range of resistance levels.

Complete Genomic Data of Burkholderia glumae Strain GX Associated with Bacterial Panicle Blight of Rice in China.

Burkholderia glumae is a seedborne pathogen causing bacterial panicle blight of rice. Here, we report the complete genome of B. glumae strain GX, which represents the first whole-genome sequence of an isolate from China. The assembled genome consisted of five contigs, with two circular chromosomes of 3,712,850 and 2,750,046 bp and three plasmids of 201,571, 105,587, and 96,100 bp. This complete genome will provide a valuable resource for further studies on bacterial panicle blight worldwide.

Rice-Associated Rhizobacteria as a Source of Secondary Metabolites against Burkholderia glumae.

Various diseases, including bacterial panicle blight (BPB) and sheath rot, threaten rice production. It has been established that Burkholderia glumae (B. glumae) is the causative agent of the above mentioned pathologies. In the present study, antagonistic activity, growth promotion, and the metabolite profiles of two rhizobacteria, isolated in different paddy fields, were assessed against B. glumae. Strains were identified based on 16S rRNA gene sequences, and the phylogenetic analyses showed that both strains belong to the genus Enterobacter, with high similarity to the strain Enterobacter tabaci NR146667.2 (99%). The antagonistic activity was assessed with the disc diffusion method. Active fractions were isolated through a liquid/liquid extraction with ethyl acetate (EtOAc) from the fermentation media, and their antibacterial activities were evaluated following the Clinical and Laboratory Standards Institute (CLSI) guidelines. The Pikovskaya modified medium was used to test the ability of in vitro inorganic phosphorus solubilization, and BSB1 proved to be the best inorganic phosphorus solubilizer, with a solubilization index (SI) of 4.5 ± 0.2. The glass-column fractionation of the EtOAc extracted from BCB11 produced an active fraction (25.9 mg) that inhibited the growth of five B. glumae strains by 85-95%. Further, metabolomic analysis, based on GC-MS, showed 3-phenylpropanoic acid (3-PPA) to be the main compound both in this fraction (46.7%), and in the BSB1 extract (28.6%). This compound showed antibacterial activity against all five strains of B. glumae with a minimum inhibitory concentration (MIC) of 1000 mg/L towards all of them. The results showed that rice rhizosphere microorganisms are a source of compounds that inhibit B. glumae growth and are promising plant growth promoters (PGP).

Heterologous production of long-chain rhamnolipids from Burkholderia glumae in Pseudomonas putida-a step forward to tailor-made rhamnolipids.

Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a ß-glycosidic bond to 3-hydroxyfatty acids with various chain lengths, and they have an enormous potential for various industrial applications. The best known native rhamnolipid producer is the human pathogen Pseudomonas aeruginosa, which produces short-chain rhamnolipids mainly consisting of a Rha-Rha-C10-C10 congener. Bacteria from the genus Burkholderia are also able to produce rhamnolipids, which are characterized by their long-chain 3-hydroxyfatty acids with a predominant Rha-Rha-C14-C14 congener. These long-chain rhamnolipids offer different physicochemical properties compared to their counterparts from P. aeruginosa making them very interesting to establish novel potential applications. However, widespread applications of rhamnolipids are still hampered by the pathogenicity of producer strains and-even more important-by the complexity of regulatory networks controlling rhamnolipid production, e.g., the so-called quorum sensing system. To overcome encountered challenges of the wild type, the responsible genes for rhamnolipid biosynthesis in Burkholderia glumae were heterologously expressed in the non-pathogenic Pseudomonas putida KT2440. Our results show that long-chain rhamnolipids from Burkholderia spec. can be produced in P. putida. Surprisingly, the heterologous expression of the genes rhlA and rhlB encoding an acyl- and a rhamnosyltransferase, respectively, resulted in the synthesis of two different mono-rhamnolipid species containing one or two 3-hydroxyfatty acid chains in equal amounts. Furthermore, mixed biosynthetic rhlAB operons with combined genes from different organisms were created to determine whether RhlA or RhlB is responsible to define the fatty acid chain lengths in rhamnolipids.

Evaluation of major Japanese rice cultivars for resistance to bacterial grain rot caused by Burkholderia glumae and identification of standard cultivars for resistance.

Bacterial grain rot (BGR), caused by the bacterial pathogen Burkholderia glumae, is one of the most destructive rice (Oryza sativa) diseases in Japan; however, there are no BGR-resistant cultivars for use in Japan. We previously developed a cut-panicle inoculation method to assess the levels of BGR resistance in the World Rice Collection (WRC). Here, we evaluated major Japanese cultivars for BGR resistance and found that none showed "strong" or "medium to strong" resistance; most were categorized as "medium to weak". On the basis of the screening results, standard cultivars for BGR resistance were selected according to resistance level and relative maturity. Our results indicate that it is necessary to introduce quantitative trait loci (QTLs) from indica or tropical japonica resistant cultivars into Japanese temperate japonica to develop BGR-resistant cultivars for Japan. We previously developed a near-isogenic line (RBG2-NIL) by introducing the genomic region containing RBG2 from 'Kele' (indica) into 'Hitomebore'. In this experiment, we confirmed the resistance level of RBG2-NIL. The resistance score of RBG2-NIL was "medium to strong", indicating its effectiveness against BGR.

Understanding the direction of evolution in Burkholderia glumae through comparative genomics.

Members of the genus Burkholderia occupy remarkably diverse niches, with genome sizes ranging from ~3.75 to 11.29 Mbp. The genome of Burkholderia glumae ranges in size from ~5.81 to 7.89 Mbp. Unlike other plant pathogenic bacteria, B. glumae can infect a wide range of monocot and dicot plants. Comparative genome analysis of B. glumae strains can provide insight into genome variation as well as differential features of whole metabolism or pathways between multiple strains of B. glumae infecting the same host. Comparative analysis of complete genomes among B. glumae BGR1, B. glumae LMG 2196, and B. glumae PG1 revealed the largest departmentalization of genes onto separate replicons in B. glumae BGR1 and considerable downsizing of the genome in B. glumae LMG 2196. In addition, the presence of large-scale evolutionary events such as rearrangement and inversion and the development of highly specialized systems were found to be related to virulence-associated features in the three B. glumae strains. This connection may explain why this bacterium broadens its host range and reinforces its interaction with hosts.

Production of p-hydroxybenzoic acid from p-coumaric acid by Burkholderia glumae BGR1.

p-Coumaric acid (pCA) is abundant in biomass with low lignin content, such as straw and stubble from rye, wheat, and barley. pCA can be isolated from biomass and used for the synthesis of aromatic hydrocarbons. Here, we report engineering of the natural pathway for conversion of pCA into p-hydroxybenzoic acid (pHBA) to increase the amount of pHBA that accumulates more than 100-fold. Burkholderia glumae strain BGR1 (BGR1) grows efficiently on pCA as a sole carbon source via a CoA-dependent non-ß-oxidation pathway. This pathway removes two carbons from pCA as acetyl-CoA yielding p-hydroxybenzaldehyde and subsequently oxidizes it to pHBA. To increase the amount of accumulated pHBA in BGR1, we first deleted two genes encoding enzymes that degrade pHBA in the ß-ketoadipate pathway. At 10 mM of pCA, the double deletion mutant BGR1_PB4 (Δphb3hΔbcl) accumulated pHBA with 95% conversion, while the control BGR1 accumulated only with 11.2% conversion. When a packed bed reactor containing immobilized BGR1_PB4 cells was operated at a dilution rate 0.2 h(-1) , the productivity of pHBA was achieved at 9.27 mg/L/h for 134 h. However, in a batch reactor at 20 mM pCA, growth of BGR1_PB4 was strongly inhibited, resulting in a low conversion of 19.3%. To further increase the amount of accumulated pCA, we identified the first enzyme in the pathway, p-hydroxcinnmaoyl-CoA synthetase II (phcs II), as the rate-limiting enzyme. Over expression of phcs II using a Palk promoter in a batch reaction at 20 mM of pCA yielded 99.0% conversion to pHBA, which is the highest concentration of pHBA ever reported using a biological process. Biotechnol. Bioeng. 2016;113: 1493-1503. © 2015 Wiley Periodicals, Inc.

Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.

Overexpression of BSR1 confers broad-spectrum resistance against two bacterial diseases and two major fungal diseases in rice.

Broad-spectrum disease resistance against two or more types of pathogen species is desirable for crop improvement. In rice, Xanthomonas oryzae pv. oryzae (Xoo), the causal bacteria of rice leaf blight, and Magnaporthe oryzae, the fungal pathogen causing rice blast, are two of the most devastating pathogens. We identified the rice BROAD-SPECTRUM RESISTANCE 1 (BSR1) gene for a BIK1-like receptor-like cytoplasmic kinase using the FOX hunting system, and demonstrated that BSR1-overexpressing (OX) rice showed strong resistance to the bacterial pathogen, Xoo and the fungal pathogen, M. oryzae. Here, we report that BSR1-OX rice showed extended resistance against two other different races of Xoo, and to at least one other race of M. oryzae. In addition, the rice showed resistance to another bacterial species, Burkholderia glumae, which causes bacterial seedling rot and bacterial grain rot, and to Cochliobolus miyabeanus, another fungal species causing brown spot. Furthermore, BSR1-OX rice showed slight resistance to rice stripe disease, a major viral disease caused by rice stripe virus. Thus, we demonstrated that BSR1-OX rice shows remarkable broad-spectrum resistance to at least two major bacterial species and two major fungal species, and slight resistance to one viral pathogen.