Enhanced resistance to seed-transmitted bacterial diseases in transgenic rice plants overproducing an oat cell-wall-bound thionin

Bacterial attack is a serious agricultural problem for growth of rice seedlings in the nursery and field. The thionins purified from seed and etiolated seedlings of barley are known to have antimicrobial activity against necrotrophic pathogens; however, we found that no endogenous rice thionin genes alone are enough for resistance to two major seed-transmitted phytopathogenic bacteria, Burkholderia plantarii and B. glumae, although rice thionin genes constitutively expressed in coleoptile, the target organ of the bacteria. Thus, we isolated thionin genes from oat, one of which was overexpressed in rice. When wild-type rice seed were germinated with these bacteria, all seedlings were wilted with severe blight. In the seedling infected with B. plantarii, bacterial staining was intensively marked around stomata and intercellular spaces. However, transgenic rice seedlings accumulating a high level of oat thionin in cell walls grew almost normally with bacterial staining only on the surface of stomata. These results indicate that the oat thionin effectively works in rice plants against bacterial attack.     

Burkholderia glumae: next major pathogen of rice?

Burkholderia glumae causes bacterial panicle blight of rice, which is an increasingly important disease problem in global rice production. Toxoflavin and lipase are known to be major virulence factors of this pathogen, and their production is dependent on the TofI/TofR quorum-sensing system, which is mediated by N-octanoyl homoserine lactone. Flagellar biogenesis and a type III secretion system are also required for full virulence of B. glumae. Bacterial panicle blight is thought to be caused by seed-borne B. glumae; however, its disease cycle is not fully understood. In spite of its economic importance, neither effective control measures for bacterial panicle blight nor rice varieties showing complete resistance to the disease are currently available. A better understanding of the molecular mechanisms underlying B. glumae virulence and of the rice defence mechanisms against the pathogen would lead to the development of better methods of disease control for bacterial panicle blight. TAXONOMY: Bacteria; Proteobacteria; Betaproteobacteria; Burkholderiales; Burkholderiaceae; Burkholderia. MICROBIOLOGICAL PROPERTIES: Gram-negative, capsulated, motile, lophotrichous flagella, pectolytic. DISEASE SYMPTOMS: Aborted seed, empty grains as a result of failure of grain filling, brown spots on panicles, seedling rot. DISEASE CONTROL: Seed sterilization, planting partially resistant lines (no completely resistant line is available). KNOWN VIRULENCE FACTORS: Toxoflavin, lipase, type III effectors.     

Constitutive expression of human lactoferrin and its N-lobe in rice plants to confer disease resistance.

The milk protein, lactoferrin, is known to have antibacterial, antiviral, and antifungal activities. To explore the possibility of conferring disease resistance in plants by expressing this protein, the gene for the full-length human lactoferrin (HLF), as well as the N-lobe, the N-terminal half molecule (HLFN), was introduced into rice plants and expressed constitutively under the control of the cauliflower mosaic virus 35S promotor. Western blot analysis of leaves from HLF-transgenic rice plants showed an 80 kDa-band, which was about 1-2 kDa less than human milk lactoferrin. HLFN was expressed as a 45-kDa protein and retained its heparin-binding property. Deglycosylation experiments suggested that both proteins produced by the plants had plant-type oligosaccharide chains. The transgenic rice plants were assessed for resistance against disease-causing bacteria, virus, and fungi. Of the pathogens tested, significant resistance against Burkholderia (Pseudomonas) plantarii, the causative agent of bacterial seedling blight disease, was observed in the transgenic plants expressing HLF or HLFN.     

Antifungal effect and mechanism of chitosan against the rice sheath blight pathogen, Rhizoctonia solani

The antifungal properties and mechanism of three types of chitosan against the rice sheath blight pathogen, Rhizoctonia solani, were evaluated. Each chitosan had strong antifungal activity against R. solani and protected rice seedlings from sheath blight, in particular, two types of acid-soluble chitosan caused a 60–91?% inhibition in mycelial growth, 31–84?% inhibition of disease incidence, and 66–91?% inhibition in lesion length. The mechanism of chitosan in protection of rice from R. solani pathogen was attributed to direct destruction of the mycelium, evidenced by scanning and transmission electron microscopic observations and pathogenicity testing; indirect induced resistance was evidenced by the changes in the activities of the defense-related phenylalanine ammonia lyase, peroxidase and polyphenol oxidase in rice seedling. To our knowledge, this is the first report on the antifungal activity of chitosan against rice R. solani.     

Quantitative Fusarium spp. and Microdochium spp. PCR assays to evaluate seed treatments for the control of Fusarium seedling blight of wheat

AIMS: To develop sensitive quantitative PCR assays for the two groups of pathogens responsible for Fusarium seedling blight in wheat: Fusarium group (Fusarium culmorum and Fusarium graminearum) and Microdochium group (Microdochium nivale and Microdochium majus); and to use the assays to assess performance of fungicide seed treatments against each group. METHODS AND RESULTS: Primers conserved between the species within each group were used to develop competitive PCR assays and used to quantify DNA of each group in wheat seed produced from inoculated field plots. Seed was used in seed treatment efficacy field experiments and the amount of DNA of each group was determined in emerged seedlings. The performance of treatments towards each group of pathogens was evaluated by comparison of the reduction in DNA in seedlings emerged from treated seed compared with untreated seed. CONCLUSIONS: DNA from the two groups of pathogens causing Fusarium seedling blight of wheat can be quantified separately using the competitive PCR assays. These assays show improved sensitivity compared with those previously reported for the individual species and allowed the quantification of pathogen DNA in seed and seedlings. Significant reductions in pathogen DNA were evident for each seed treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantification of DNA for each group allows the evaluation of seed treatment performance towards the two components of Fusarium seedling blight disease complex. The approach taken and the assays developed in this study will be of use for the study of other Fusarium disease complexes and their control. Based on the results reported here on the seedling stage of crop development, further studies that examine the control of seed-borne pathogens through fungicide seed treatments throughout the growing season are warranted.     

SEEDLING BLIGHT OF SUGAR-CANE-A NEW DISEASE CAUSED BY HELMINTHOSPORIUM SACCHARI BUTLER

The fungus Helminthosporium sacchari Butler, known to be the cause of eye spot, a common air-borne disease of sugar-cane leaves, is now shown to be the cause of an undescribed seed-borne disease of sugar-cane seedlings. It is therefore suggested that the disease should be renamed eye spot and seedling blight of sugarcane.
The symptoms of seedling blight are described, and the pathogenicity of the fungus to sugar-cane seedlings is confirmed. Incidence of seedling blight is shown to be markedly affected by the growing conditions, and disease-escape is common. The two most important factors predisposing seedlings to attack appear to be (i) the relative humidity of the atmosphere, and (ii) the occurrence of a check to the continuous growth of the seedlings.
With the discovery of the existence of seedling blight, eye spot assumes a particular significance in plant-breeding plots, because it may be responsible for the loss of sugar-cane seedlings, any one of which is potentially a desirable new variety.     

一株水稻纹枯菌拮抗细菌的分离与鉴定

【目的】从土壤中分离并鉴定水稻纹枯菌拮抗细菌,测定其体外抑菌和温室防治效果。【方法】采用系列稀释法和平板对峙法筛选拮抗细菌,基于形态、生理特征及16S rDNA序列鉴定其分类地位,采用种子细菌化温室试验测定其防效。【结果】从蔬菜根际土壤中筛选出一株纹枯菌拮抗细菌,命名为kwkjT4。菌株具有明显的体外抑菌活性,对水稻纹枯病的温室防效与井冈霉素相当,初步鉴定为假紫色色杆菌(Chromobacterium pseudoviolaceum)。最适生长条件为pH 7.0,温度32°C,培养时间为36 h;抑菌活性物质产生的最适培养条件为pH 6.0,温度28°C,培养时间为48 h;表明两者并不一致。【结论】kwkjT4菌株在水稻纹枯病的生物防治中具有潜在的应用价值。这是C.pseudoviolaceum拮抗纹枯菌的首次报道。     

Distribution and Races of Xanthomonas axonopodis pv. malvacearum on Cotton (Gossypium hirsutum) in Uganda

Surveys in 1995 and 1996 showed that bacterial blight caused by Xanthomonas axonopodis pv. malvacearum occurs throughout the main cotton growing areas of Uganda, causing seedling blight, angular leaf spot and bacterial boll rot. During the vegetative and early fruiting stages of crop growth, severe symptoms of `blackarm' spread from leaves to the stem, causing loss of fruiting branches. A set of Upland cotton cultivars ( Gossypium hirsutum ) were then used to determine the races of the blight bacterium present in Uganda. Many of the isolates induced moderate to severe symptoms on all the test hosts except 101–102B, indicating infection with race 10 or 18. The next most common isolate was race 7. Races 16 and 6 were also identified and 23% of isolates caused symptoms on all the differential cultivars including 101–102B, results indicating the presence of a race of the pathogen which may be the same as that identified in countries neighbouring Uganda and designated as race 20.     

Identification of major quantitative trait loci qSBR11-1 for sheath blight resistance in rice

Sheath blight caused by Rhizoctonia solani Kühn is one of the important diseases of rice, resulting in heavy yield loss in rice every year. No rice line resistant to sheath blight has been identified till date. However, in some rice lines a high degree of resistance to R. solani has been observed. An indica rice line, Tetep, is a well documented source of durable and broad spectrum resistance to rice blast as well as quantitative resistance to sheath blight. The present study identified genetic loci for quantitative resistance to sheath blight in rice line Tetep. A mapping population consisting of 127 recombinant inbred lines derived from a cross between rice cultivars HP2216 (susceptible) and Tetep (resistant to sheath blight) was evaluated for sheath blight resistance and other agronomic traits for 4 years across three locations. Based on sheath blight phenotypes and genetic map with 126 evenly distributed molecular markers, a quantitative trait loci (QTLs) contributing to sheath blight resistance was identified on long arm of chromosome 11. Two QTL mapping approaches i.e., single marker analysis and composite interval mapping in multi environments were used to identify QTLs for sheath blight resistance and agronomical traits. The QTL qSBR11-1 for sheath blight resistance was identified between the marker interval RM1233 (26.45 Mb) to sbq33 (28.35 Mb) on chromosome 11. This region was further narrowed down to marker interval K39516 to sbq33 (~0.85 Mb) and a total of 154 genes were predicted including 11 tandem repeats of chitinase genes which may be responsible for sheath blight resistance in rice line Tetep. A set of 96 varieties and a F₂ population were used for validation of markers linked to the QTL region. The results indicate that there is very high genetic variation among varieties at this locus, which can serve as a starting point for allele mining of sheath blight resistance.     

Effect of volatile substances of Streptomyces platensis F-1 on control of plant fungal diseases

Headspace volatile substances (VS) produced by Streptomyces platensis F-1 were preliminarily identified using GC–MS. The effects of VS released by S. platensis F-1 on the control of leaf blight/seedling blight of rice caused by Rhizoctonia solani, leaf blight of oilseed rape caused by Sclerotinia sclerotiorum and fruit rot of strawberry caused by Botrytis cinerea, as well as on the growth of these three pathogenic fungi, were investigated. Results showed that sixteen volatile compounds were tentatively identified in 1-week-old cultures of S. platensis F-1 grown on autoclaved wheat seeds. They could be chemically grouped into alcohols, esters, acids, alkanes, ketones and alkenes. The most abundant composition in volatiles of S. platensis F-1 is geosmin, an earthy-muddy–smelling compound. Two antifungal compounds, phenylethyl alcohol and (+)-epi-bicyclesesquiphellandrene, were detected in the volatile profile of S. platensis F-1. Consistent fumigation of healthy tissues of rice, oilseed rape and strawberry to VS of S. platensis effectively reduced the incidence and/or the severity of leaf blight/seedling blight of rice (R. solani), leaf blight of oilseed rape (S. sclerotiorum) and fruit rot of strawberry (B. cinerea). A significant (P < 0.05) suppression of the mycelial growth of R. solani, S. sclerotiorum and B. cinerea by the VS of S. platensis was observed. The potential of using VS of S. platensis F-1 as a biofumigant to control plant fungal diseases is discussed.     

Comparative genome analysis of rice-pathogenic Burkholderia provides insight into capacity to adapt to different environments and hosts

Background

In addition to human and animal diseases, bacteria of the genus Burkholderia can cause plant diseases. The representative species of rice-pathogenic Burkholderia are Burkholderia glumae, B. gladioli, and B. plantarii, which primarily cause grain rot, sheath rot, and seedling blight, respectively, resulting in severe reductions in rice production. Though Burkholderia rice pathogens cause problems in rice-growing countries, comprehensive studies of these rice-pathogenic species aiming to control Burkholderia-mediated diseases are only in the early stages.

Results

We first sequenced the complete genome of B. plantarii ATCC 43733^T. Second, we conducted comparative analysis of the newly sequenced B. plantarii ATCC 43733^T genome with eleven complete or draft genomes of B. glumae and B. gladioli strains. Furthermore, we compared the genome of three rice Burkholderia pathogens with those of other Burkholderia species such as those found in environmental habitats and those known as animal/human pathogens. These B. glumae, B. gladioli, and B. plantarii strains have unique genes involved in toxoflavin or tropolone toxin production and the clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bacterial immune system. Although the genome of B. plantarii ATCC 43733^T has many common features with those of B. glumae and B. gladioli, this B. plantarii strain has several unique features, including quorum sensing and CRISPR/CRISPR-associated protein (Cas) systems.

Conclusions

The complete genome sequence of B. plantarii ATCC 43733^T and publicly available genomes of B. glumae BGR1 and B. gladioli BSR3 enabled comprehensive comparative genome analyses among three rice-pathogenic Burkholderia species responsible for tissue rotting and seedling blight. Our results suggest that B. glumae has evolved rapidly, or has undergone rapid genome rearrangements or deletions, in response to the hosts. It also, clarifies the unique features of rice pathogenic Burkholderia species relative to other animal and human Burkholderia species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1558-5) contains supplementary material, which is available to authorized users.     

Infection Process of Burkholderia glumae Before Booting Stage of Rice

Burkholderia glumae is a well‐known pathogen for causing bacterial panicle blight of rice. In this study, the infection process of B. glumae in rice plants at different growing stages was tracked by means of real‐time fluorescence quantitative PCR. Burkholderia glumae tended to colonize at the growing point of rice plants, and the biomass of population was 10⁴ to 10⁸ CFU/g. The most intensive colonization was detected in the upmost leaf in the two‐leaf period. However, after the two‐leaf period, the population of pathogens decreased significantly, and they successfully recovered in the booting stage and broke out in panicles. We also illustrated the incubation location of B. glumae by presenting the infection pattern in the seedling and tillering stage of rice. Under fluorescent microscopy, the gfp‐labelled pathogens were first found in the vascular bundle of lateral roots, taproots and injured cells, then they were observed in the root hairs, epidermal cells and main root cap. The pathogens in the vascular bundle laterally dispersed towards the epidermal cells. By spray application of a bacterial suspension, the pathogens landed on the leaf sheaths and leaves, colonized in the epidermal hairs and leaf hairs, or invaded into the cells through the stomas. At the same time, the pathogens from the vascular bundle of the roots spread into the vascular bundle of leaf sheaths and leaves, which caused the leaves to curl and wilt, beginning from the tip.     

Characterisation,screening and selection of Bacillus subtilis isolates for its biocontrol efficiency against major rice diseases

A Soil bacterium, Bacillus subtilis, isolated from the rhizosphere of rice, showed high biocontrol activity against blast, sheath blight and bacterial blight. In the present study, four B. subtilis strains isolated from paddy soil were studied under laboratory, greenhouse and field conditions. Among the four strains assayed, UASP17 gave maximum inhibition of paddy pathogens and was validated under field trials. B. subtilis (UASP17) under different doses and methods of applications was evaluated for two seasons at Agriculture Research Station (UAS, Raichur). UASP17 was effective in reducing the severity of blast (9.00% and 15.57%), bacterial leaf blight (BLB) (5.00% and 6.11%) and sheath blight (11.93% and 4.17%) diseases for two seasons. The application of bioagent also increased the paddy grain yield (61.00 and 64.30?Q/ha) in the two seasons, respectively. Taken together, these results indicate that B. subtilis UASP17 as seedling dip for 30?min (10?mL/L of water) prior to transplanting and 2.50?L/ha foliar application was effective in managing the diseases of paddy.     

Isolation and identification of a potent allelopathic substance in rice root exudates

A search for growth inhibitors in rice root exudates was undertaken in order to clarify the allelopathic system in rice ( Oryza sativa L.). Rice seedlings inhibited the growth of cress ( Lepidium sativum L.) and lettuce ( Lactuca sativa L.) seedlings when the cress and lettuce were grown with rice seedlings. The putative compound causing the inhibitory effect of rice seedlings was isolated from their culture solution, and the chemical structure of the inhibitor was determined by spectral data as momilactone B. Momilactone B inhibited the growth of cress and lettuce seedlings at concentrations greater than 3 and 30 µ M , respectively. The concentration of momilactone B was 3.4 and 1.1 nmol per seedling in the culture solutions of husked and non-husked rice seedlings, respectively. These results suggest that rice seedlings may release momilactone B into the environment and the stress caused by the husk-treatment may increase the amount of momilactone B released. Thus, momilactone B may play an important role in rice allelopathy.     

Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

Diterpene Phytoalexins Are Biosynthesized in and Exuded from the Roots of Rice Seedlings

Rice (Oryza sativa L.) produces a variety of diterpene phytoalexins, such as momilactones, phytocassanes, and oryzalexins. Momilactone B was previously identified as an allelopathic substance exuded from the roots of rice. We identified in this present study momilactone A and phytocassanes A–E in extracts of, and exudates from, the roots of rice seedlings. The concentration of each compound was of the same order of magnitude as that of momilactone B. Expression analyses of the diterpene cyclase genes responsible for the biosynthesis of momilactones and phytocassanes suggest that these phytoalexins found in roots are primarily biosynthesized in those roots. None of phytocassanes B–E exhibited allelopathic activity against dicot seedling growth, whereas momilactone A showed much weaker allelopathic activity than momilactone B. The exudation of diterpene phytoalexins from the roots might be part of a system for defense against root-infecting pathogens.     

Antimicrobial compounds profile during Cheonggukjang fermentation against Xanthomonas oryzae pv. oryzae (Xoo)

Xanthomonas oryzae causes rice bacterial blight, which has been reported as one of the most destructive diseases of rice. Metabolites were identified through cheonggukjang, a traditional Korean fermented soybean product fermented by the Bacillus spp., to control the bacteria. HPLC, MS, and UPLC-Q-TOF-MS analyses were performed to identify metabolites responsible for antimicrobial activity. In this analysis, the m/z values of 253.0498, 283.0600, 269.0455, 992.6287, and 1,006.6436 were identified as daidzein, glycitein, genistein, surfactin B, and surfactin A, respectively. The levels of surfactin B and surfactin A were found to be high at 24 h (4.35 μg/ml) and 36 h (3.43 μg/ml) of fermentation, respectively.     

Morphological,pathological and molecular characterisation of rice sheath blight disease causal organism Rhizoctonia solani AG-1 IA in Egypt

Rice sheath blight disease caused by Rhizoctonia solani is considered a distractive soil-borne disease of rice production worldwide. The study aimed to determine the causal organism of sheath blight symptoms in Egyptian rice fields. Sheath blight symptoms were first observed in a small area during 2013, 2014 and 2016 seasons, later in a wide area of rice fields in 2016 to 2018 seasons. Pathogen identification was carried out based on morphological traits and internal transcribed spacer sequencing. Thirty-six isolates were identified as R. solani fungus. The isolates exhibited a wide range of variability in their morphological traits and virulence patterns. Five isolates were sequenced and aligned with Chinese isolates with 75–100% identity. This is the first report of R. solani AG-1 IA that associated with rice sheath blight in Egypt. Initiate a breeding program for disease resistance and integrated disease management procedures are important to keep the disease under control.     

Biological control of rice bacterial blight by plant-associated bacteria producing 2,4-diacetylphloroglucinol

Certain plant-associated strains of fluorescent Pseudomonas spp. are known to produce the antimicrobial antibiotic 2,4-diacetylphloroglucinol (DAPG). It has antibacterial, antifungal, antiviral, and antihelminthic properties and has played a significant role in the biological control of tobacco, wheat, and sugar beet diseases. It has never been reported from India and has not been implicated in the biological suppression of a major disease of the rice crop. Here, we report that a subpopulation of 27 strains of plant-associated Pseudomonas fluorescens screened in a batch of 278 strains of fluorescent pseudomonads produced DAPG. The DAPG production was detected by a PCR-based screening method that used primers Phl2a and Phl2b and amplified a 745-bp fragment characteristic of DAPG. HPLC, 1H NMR, and IR analyses provided further evidence for its production. We report also that this compound inhibited the growth of the devastating rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae in laboratory assays and suppressed rice bacterial blight up to 59%-64% in net-house and field experiments. Tn5 mutants defective in DAPG production (Phl-) of P. fluorescens PTB 9 were much less effective in their suppression of rice bacterial blight.