Identification of QTLs underlying resistance to a virulent strain of Xanthomonas oryzae pv. oryzae in rice cultivar DV85

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice in China. A strongly virulent Xoo strain, designated Z-173, is widely distributed across China and Southeast Asia. Indica rice DV85 is known to carry the two resistance genes, xa5 and Xa7. However, their effectiveness against Z-173 is unknown. Using a recombinant inbred line (RIL) population derived from a cross between DV85 and the susceptible cultivar Kinmaze, we have identified the quantitative trait loci (QTLs) responsible for the resistance of DV85 to Z-173. Following 2 years of phenotyping, three QTLs associated with the resistance were detected. These were linked to RFLP markers X362, X292 and G1091 on chromosomes 3, 5, and 6, respectively. Qxa-5 and Qxa-6 probably correspond to xa5 and Xa7, respectively. Both the xa5 and Xa7 resistances are stable over different years, and act independently of one another in determining resistance. The effect of xa5 was larger than that of Xa7. Efficient ways to improve the resistance to Z-173 are discussed.     

Biological control of tomato bacterial speck using Punica granatum fruit peel extract

Pseudomonas syringae pv. tomato, the causal agent of tomato bacterial speck, is common in greenhouses and fields all over Italy. The antibacterial activity of ethanol extracts of Punica granatum (pomegranate) fruit peels on P. syringae pv. tomato was studied both in vitro and in vivo. The minimum in vitro inhibition value for peel extract of P. granatum on the P. syringae pv. tomato Pt 1301 strain (from Turkey) was obtained at a concentration of 0.5%. The in vivo antibacterial action of this natural substance lasted at least 15 days, permitting the replacement or reduction of the commonly used copper compounds. The active components of the pomegranate peel extracts were ellagic and gallic acids.     

Pseudomonas aeruginosa produces secondary metabolites that have biological activity against plant pathogenic Xanthomonas species

The bioactivity of compounds produced by Pseudomonas aeruginosa (LN strain), against three Xanthomonas species was investigated under greenhouse conditions and using electron microscopy. Chromatographic fractions EAP, VLC3, VLC4, VLC3d and VLC4f were tested against Xanthomonas axonopodis pv. malvacearum, X. axonopodis pv. phaseoli and X. axonopodis pv. citri by disc diffusion. Fractions with antibiotic activity were tested in vivo under greenhouse conditions and their bioactivity was evaluated. Scanning electron microscopy showed that VLC4f affects biofilm formation while VLC4f and VLC3d both affect cell morphology. The semi-purified fractions controlled bacterial diseases caused by Xanthomonas spp. when sprayed on plants under greenhouse conditions. The VLC4f fraction showed superior results in disease management, reducing the number of lesions on cotton and orange leaves by 94%, and reducing disease severity in bean leaves by 73%. The data suggest that the fractions were effective and have potential as an alternative to conventional bactericides in the control of plant diseases caused by Xanthomonas sp under greenhouse conditions.     

Resistance responses of rice to rice blast fungus after seed treatment with the endophytic Achromobacter xylosoxidans AUM54 strains

In the present investigation, we studied resistance imparted by seed treatment with an endophytic strain of Achromobacter xylosoxidans, AUM54, against rice blast caused by Magnaporthe oryzae. In vitro studies showed that A. xylosoxidans AUM54 was able to inhibit mycelial growth of M. oryzae by 11% and was able to increase rice germination and seedling vigor index of rice by 31 and 114%, respectively. AUM54 also showed better survivability in the spermosphere and spermoplane and was able to move systemically through the roots and stem. Among the evaluated carriers, liquid formulation amended with 2% glycerol sustained the maximum bacterial population (7.4 log cfu ml⁻¹) after six-months-storage at room temperature. Plants treated with A. xylosoxidans AUM54 followed by inoculation with M. oryzae showed a significant increase in the activities of defense related enzymes such as polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia-lyase (PAL) and chitinase. A. xylosoxidans AUM54 treatment was able to reduce blast disease incidence by 39% in treated rice plants. Additionally, inoculation with A. xylosoxidans AUM54 significantly enhanced the growth (3–13% plant height), and yield (11–31%) of inoculated rice plants under no-disease and disease conditions in the greenhouse experiments.     

Rhizosphere Bacteria for Biocontrol of Bacterial Blight and Growth Promotion of Rice

Several bacterial strains were isolated from different rhizospheres. Among these, strain PDY7 exhibited strong antibacterial activity against the rice bacterial blight (BB) pathogen Xanthomonas oryzae pv. oryzae (Xoo) by the laboratory dual plate assays. The antibacterial property of the strain PDY7 was further investigated for the production of 2,4-diacetylphloroglucinol (DAPG), which amplified a characteristic of 629-bp DNA fragment by PCR-based screening method using phlD primers. The application of phlD positive strains was carefully evaluated for disease control and growth promotion of rice plants under field conditions. The selected strain PDY7 suppressed the rice BB by 58.83% and 51.88% under glass house and field conditions, respectively. In addition, the strain PDY7 showed significant two-fold increase in root length (18.08 cm), shoot length (29.81 cm), and grain yield (96.07 g). Strain PDY7 promoted the growth of rice plants by production of indole-3-acetic acid (IAA), which was determined by high performance liquid chromatography (HPLC) analysis. Our findings suggest that PDY7 belongs to the P. fluorescens group and can serve as potential biocontrol of BB as well as biofertilizer agent for growth promotion of rice.     

Occurrence of rice blast (Magnaporthe oryzae) and identification of potential resistance sources in Uganda

Rice blast caused by Magnaporthe oryzae continues to be the most destructive disease of rice worldwide, and is a number one disease of rice in Uganda. We present the footprints of M. oryzae importance, distribution, incidence and severity in the rice growing agro-ecologies of Uganda for 2009/2010, and the potential mitigation measures. Our data show that rice blast affects more than 50% of the cultivated rice area on average, and ranks as the most important disease encountered in the field. Between and within agroecologies, both incidence and severity varied (P < 0.05) reflecting the contribution of different cropping practices on rice blast epiphytotics. The highest blast incidence and severities were recorded in Bugiri, Butaleja, Mbale and Lira farmlands, which are the ancestral rice cultivation areas in Uganda. These areas showed mean yield reductions of >30% relative to other locations, suggesting that rice production history played a significant role in rice blast outbreaks. Broadcasting and drill seeding yielded 42.4% less than transplanted rice. Growing two rice crops per year is one of the methods suggested to increase rice production in Uganda, but there was a higher disease incidence in the late season than in the early season, indicating the need for improved resistant varieties. Four blast resistance genes (Pi9, Piz-t, Pi19 and Piz-5) and the cultivar Tetep had the lowest (≤4) blast severity scores in all the test locations. It could be suggested that these genes are potential resistance sources for developing varieties, which would be more relevant for the double cropping systems.     

Bacterial Impact on H2O2 Accumulation during the Interaction between Xanthomonas and Rice

Abstract

Localization of hydrogen peroxide (H₂O₂) accumulation during the interaction between rice and Xanthomonas oryzae pv. oryzae (X. oryzae pv. oryzae) was observed by histochemical analysis and electron microscopy. The changes that occurred in an avrXa7 mutantstrain of X. oryzae pv. oryzae including the decreased production of endogenous H₂O₂, impacted on the mean level of H₂O₂ accumulation during the interaction with the plant. The results of catalase and aminotriazole treatments indicated that the changes of H₂O₂ accumulation during the interaction are induced by the impairment of endogenous H₂O₂ accumulation in X. oryzae pv. oryzae. These results suggested that bacterial pathogen is a potential source of the H₂O₂ accumulated in the interaction between rice and X. oryzae pv. oryzae.     

Induction of lignin and pathogenesis related proteins in dragon fruit plants in response to submicron chitosan dispersions

Chitosan is capable of triggering the induction of defence responses of the plants. This study was carried out to investigate in vitro and in vivo antifungal activity of submicron chitosan dispersions and also to determine the mechanism of induced resistance in dragon fruit plants by strengthening of cell wall and production of pathogenesis related proteins. Highest inhibition in radial mycelial growth (90.2%) was obtained by 600 nm droplet size at 1.0% chitosan concentration. None of the emulsifiers showed any antifungal effect during in vitro studies. Field studies showed the reduction of 22% disease incidence and 66% disease severity in anthracnose on dragon fruit plants treated with 600 nm droplet size as compared to control plants after one month of assessment. Lignin contents in cell wall of dragon fruit plants increased 2 folds in plants treated with 1.0% chitosan at 600 nm droplet size while 11 and 10 folds increase in β-1,3-glucanase and chitinase activity, respectively was also recorded in the plants inoculated with Colletotrichum gloeosporioides. The accumulation of lignin and higher level activity of pathogenesis related proteins in response to submicron chitosan dispersions may bolster in mounting effective resistance against the devastating disease of anthracnose.     

Antifungal activity of Ziziphora clinopodioides Lam. essential oil against Sclerotinia sclerotiorum on rapeseed plants (Brassica campestris L.)

The present study aimed to determine the in vitro and in vivo effects of Ziziphora clinopodioides Lam. essential oil (ZCEO) against Sclerotinia sclerotiorum on rapeseed plants. Gas chromatography-mass spectrometry (GC-MS) detected 21 compounds that represented 98.3% of the total amount of extracted oil, which mainly comprised pulegone (53.5%), isomenthone (10.4%), and carvone (5.7%). The mycelial growth of S. sclerotiorum was completely inhibited at essential oil concentrations of 1.25 and 0.15 μl ml⁻¹ under contact and vapor phase conditions, respectively. Under contact phase, the germination of sclerotial was inhibited at the concentration of 1.00 μl ml⁻¹. The essential oil concentration of 0.15 μl ml⁻¹ in the vapor phase showed a strong inhibitory effect on sclerotial germination. Sclerotinia sclerotiorum growth on detached rapeseed leaves and potted rapeseed plants were dose dependently inhibited by the essential oil. Considerable morphological changes were also observed in the fungal hyphae and sclerotia. Both in vitro and in vivo results indicated ZCEO can effectively inhibit the growth of S. sclerotiorum. Thus, ZCEO could be used for crop protection.     

Genetic Improvement of Rice for Bacterial Blight Resistance: Present Status and Future Prospects

The production and productivity of rice has been challenged due to biotic and abiotic factors. Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae, is one of the important biotic stress factors, which reduces rice production by 20%–50%. The deployment of host plant resistance is the most preferred strategy for management of BB disease, and breeding disease resistant varieties remains a very economical and effective option. However, it is difficult to develop rice varieties with durable broad-spectrum resistance against BB using conventional approaches alone. Modern biotechnological tools, particularly the deployment of molecular markers, have facilitated the cloning, characterization and introgression of BB resistance genes into elite varieties. At least 46 BB resistance genes have been identified and mapped from diverse sources till date. Among these, 11 genes have been cloned and characterized. Marker-assisted breeding remains the most efficient approach to improve BB resistance by introducing two or more resistance genes into target varieties. Among the identified genes, xa5, xa13 and Xa21 are being widely used in marker-assisted breeding and more than 70 rice varieties or hybrid rice parental lines have been improved for their BB resistance alone or in combination with genes/QTLs conferring tolerance to other stress. We review the developments related to identification and utilization of various resistance genes to develop BB resistant rice varieties through marker-assisted breeding.     

Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79

Strain BC79, isolated from primeval forest soil in Qinling, Mountains, China, was identified as Bacillus methylotrophicus based on morphological, biochemical, physiological and chemotaxonomic analyses as well as phylogenetic 16S rDNA sequencing data. This strain was able to suppress mycelial growth and conidial germination of numerous plant pathogenic fungi in dual cultures on solid media. For exploring potential biocontrol activity, we assessed fermentation conditions for studying B. meth1ylotrophicus BC79. The active substance of BC79, phenaminomethylacetic acid, was extracted by TLC and HPLC, and identified as the strongest inhibitory substance described in B. methylotrophicus. Experiments in a greenhouse showed that application of BC79 culture filtrates 24 h before inoculation of Magnaporthe oryzae, the causal agent of rice blast, had 89.87% biocontrol efficiency. B. methylotrophicus BC79 colonized rice plant tissues and at 10 days after filtrate application, its population in leaves (1.65 × 10⁸ CFU/g) was much larger than in stems (6.78 × 10⁷ CFU/g) or roots (3.56 × 10⁷ CFU/g). Field trials indicated that BC79 culture filtrate (4000 g/667 m²) showed the highest efficiency for M. oryzae, with 84.8% biocontrol effect, followed by of 15% phenaminomethylacetic acid extract (75.5%) and 20% tricyclazole (76.1%). Seedling and post-transplant stages were the best periods to apply BC79 for control of rice blast. The B. methylotrophicus BC79 strain hence has enormous potential as an agricultural agent for biocontrol of rice blast.     

Improvement of Bacterial Blight Resistance in Rice Cultivars Jyothi and IR50 via Marker-Assisted Backcross Breeding

Abstract

In pathogen population analysis of 208 Xanthomonas oryzae pv. oryzae(Xoo) strains that were assembled from different parts of India, 21 pathotypes were identified on the basis of disease reactions on near-isogenic lines (NILs) and 13 pathotypes, on rice differentials. Rice cultivars, Jyothi and IR50, which are high yielding but highly prone to bacterial blight (BB) caused by pathogen populations of Xanthomonas oryzae pv. oryzae in India, were chosen. To improve the BB resistance of these two varieties, a pyramid line, NH56, containing four R-genes, Xa4, xa5, xa13, and Xa21, was selected as the R-donor based on resistance to existing pathogen population. The four R-genes were successfully transferred to cultivars through a traditional backcross method and their presence was documented with marker-aided selection (MAS). Thirty BC₄F₂ plants derived from JxNH56 (cv. Jyothi) and 45 BC₄F₂ plants derived from IR50xNH56 (cv. IR50) had all four resistance genes (Xa4, xa5, xa13, and Xa21), which should be useful resistance donors for breeding other BB-resistant elite indica varieties.     

Efficacy of Lawsonia inermis leaves extract and its phenolic compounds against olive knot and crown gall diseases

This study was undertaken to determine the antibacterial efficacy of the leaves extracts of Lawsonia inermis in vitro and in vivo, against the phytopathogenic bacteria Pseudomonas savastanoi pv savastanoi IVIA 1628 and Agrobacterium tumefaciens. The hydroalcoholic extract of L. inermis was fractionated by liquid–liquid partition using hexane, chloroform, ethyl acetate and butanol. Among the tested fractions, the ethyl acetate (EtOAcF) and chloroform (CHCl₃F) extracts exhibited high inhibition against all tested plant pathogens. The extracts exerted a bactericidal activity against both P. savastanoi and A. tumefaciens with MIC values ranging from 1.25 to 2.5 mg/ml. In planta experiments, the EtOAcF, tested at four concentrations (0.4, 0.2, 0.1 and 0.05 mg/wounds), completely inhibited the formation of knots on twigs of olive and tomato plants inoculated with pathogenic strains of P. savastanoi and A. tumefaciens (strains B6 and C58). The phytochemical screening revealed that the L. inermis fractions contain flavonoids, quinones, tannins and terpenoids. The qualitative analysis of the EtOAcF by LC–DAD–ELSD–ESI/MSⁿ showed the presence of eight identified phenolic and phenolic glycoside compounds. These results suggest that L. inermis extracts could be used to control plant bacterial diseases caused by P. savastanoi and A. tumefaciens.     

Transgene Stacking and Coordinated Expression of Plant Defensins Confer Fungal Resistance in Rice

Transgenic rice with cleavable chimeric polyprotein and single-protein gene constructs placed under the control of single maize ubiquitin promoter were generated by Agrobacterium tumefaciens-mediated transformation. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), Dm-AMP1 and Rs-AFP2, from the seeds of Dahlia merckii and Raphanus sativus, respectively. These genes were linked by a 16 amino acid Ib-AMP linker peptide region isolated from the seeds of Impatiens balsamina. Average expression of Dm-AMP1 and Rs-AFP2 was 8.35?±?0.56 and 8.1?±?0.6 2 g/mg of total soluble proteins in the leaf extracts of transgenic plants. Plants transformed with polyprotein construct showed significantly improved disease resistance against Magnaporthe oryzae and Rhizoctonia solani by 90% and 79%, respectively, as compared to untransformed plants. The plants transformed with polyprotein construct were more resistant to M. oryzae compared to plants transformed with control single-gene constructs of Dm-AMP1 and Rs-AFP2. The chimeric polyprotein was cleaved and present in equimolar concentration in transgenic rice and the individual AMPs were secreted into the extracellular space. The results of in vitro, in planta, and microscopic analyses suggest that chimeric polyprotein expression has the potential to provide broad-spectrum disease resistance in rice.     

RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae,a Causative Agent for Bacterial Leaf Blight Disease in Rice

Xanthomonas oryzae pv. oryzae (Xoo) is a widespread pathogen causing bacterial leaf blight (BLB) disease, devastating rice productivity in many cultivated areas of Thailand. A specific and simple method for Xoo detection is required to improve surveillance of disease transmission and outbreak. This study developed a recombinase polymerase amplification (RPA) assay assisted with CRISPR-cas12a assay (RAC) for Xoo detection from bacterial cell suspension of infected rice samples without DNA extraction. The efficiency of the RAC system for Xoo detection using either Xoo80 or Xoo4009 locus was optimized to amplify and determine the sensitivity and specificity using a Xoo DNA template from bacterial cell suspension of infected rice samples without DNA extraction. The RAC system using the Xoo4009 locus gave a higher specificity than Xoo80 locus, because only Xoo species was amplified positive RPA product with fluorescence signal by cas12a digestion, which indicated no cross reactivity. Optimal RAC using the Xoo4009 locus enabled diagnosis of Xoo presence from both plant extracted samples of Xoo artificially inoculated rice leaves within 3 d post-inoculation without symptomatic BLB appearance, and Xoo naturally infected rice. Findings exhibited that RAC using the Xoo4009 locus offered sensitivity, specificity and simplicity for Xoo detection, with low intensities of Xoo-DNA (1 × 10³ copies/μL) and Xoo-cell (2.5 × 10³ cfu/mL). This developed RAC system showed significantly potential for Xoo detection at point-of-care application for early signs of BLB disease outbreak in rice fields.     

Marker-Assisted Selection of Xa21 Conferring Resistance to Bacterial Leaf Blight in indica Rice Cultivar LT2

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive diseases in Asian rice fields. A high-quality rice variety, LT2, was used as the recipient parent. IRBB21, which carries the Xa21 gene, was used as the donor parent. The resistance gene Xa21 was introduced into LT2 by marker-assisted backcrossing. Three Xoo races were used to inoculate the improved lines following the clipping method. Eleven BC₃F₃ lines carrying Xa21 were obtained based on molecular markers and agronomic performance. The 11 lines were then inoculated with the three Xoo races. All the 11 improved lines showed better resistance to BLB than the recipient parent LT2. Based on the level of resistance to BLB and their agronomic performance, five lines (BC₃F₃ 5.1.5.1, BC₃F₃ 5.1.5.12, BC₃F₃ 8.5.6.44, BC₃F₃ 9.5.4.1 and BC₃F₃ 9.5.4.23) were selected as the most promising for commercial release. These improved lines could contribute to rice production in terms of food security.