<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: a biocontrol agent against <Emphasis Type="Italic">Bipolaris oryzae</Emphasis>

Rice brown spot, caused by Bipolaris oryzae, can be a serious disease causing a considerable yield loss. Trichoderma harzianum is an effective biocontrol agent for a number of plant fungal diseases. Thus, this research was carried out to investigate the mechanisms of action by which T. harzianum antagonizes Bipolaris oryzae in vitro, and the efficacy of spray application of a spore suspension of T. harzianum for control of rice brown spot disease under field conditions. In vitro, the antagonistic behavior of T. harzianum resulted in the overgrowth of B. oryzae by T. harzianum, while the␣antifungal metabolites of T.␣harzianum completely prevented the linear growth of B. oryzae. Light and scanning electron microscope (SEM) observations showed no evidence that mycoparasitism contributed to the aggressive nature of the tested isolate of T. harzianum against B. oryzae. Under field conditions, spraying of a spore suspension of T. harzianum at 10⁸ spore ml⁻¹ significantly reduced the disease severity (DS) and disease incidence (DI) on the plant leaves, and also significantly increased the grain yield, total grain carbohydrate, and protein, and led to a significant increase in the total photosynthetic pigments (chlorophyll a and b and carotenoids) in rice leaves.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

<Emphasis Type="Italic">In planta</Emphasis> gene expression analysis of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pathovar <Emphasis Type="Italic">oryzae</Emphasis>, African strain MAI1

Background  

Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian X. oryzae pv. oryzicola (Xoc).     

Fine genetic mapping of <Emphasis Type="Italic">xa24</Emphasis>, a recessive gene for resistance against <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating plant bacterial disease worldwide. Different bacterial blight resistance (R) genes confer race-specific resistance to different strains of Xoo. We fine mapped a fully recessive gene, xa24, for bacterial blight resistance to a 71-kb DNA fragment in the long arm of rice chromosome 2 using polymerase chain reaction-based molecular markers. The xa24 gene confers disease resistance at the seedling and adult stages. It mediates resistance to at least the Philippine Xoo races 4, 6 and 10 and Chinese Xoo strains Zhe173, JL691 and KS-1-21. Sequence analysis of the DNA fragment harboring the dominant (susceptible) allele of xa24 suggests that this gene should encode a novel protein that is not homologous to any known R proteins. These results will greatly facilitate the isolation and characterization of xa24. The markers will be convenient tools for marker-assisted selection of xa24 in breeding programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and Characterization of a Recombinant Bacteriophytochrome of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pathovar <Emphasis Type="Italic">oryzae</Emphasis>

Phytochrome-like proteins have been recently identified in prokaryotes but their features and functions are not clear. We cloned a gene encoding the phytochrome-like protein (XoBphP) in a pathogenic bacteria, Xanthomonas oryzae pv. oryzae (Xoo) and investigated characteristics of the protein using a recombinant XoBphP. The N-terminal region of XoBphP containing the PAS/GAF/PHY domains is highly similar to most bacteriophytochromes, but Cys4, corresponding to Cys24 of DrBphP, isn’t involved in chromophore attachment. Recombinant XoBphP could bind a bilin molecule and a differential spectrum from Pr/Pfr shows that XoBphP has similar characteristics of known bacteriophytochromes with shifted absorption maxima of 683 and 757 nm for the Pr and Pfr forms. Unlike other bacteriophytochromes, XoBphP has no histidine kinase domain at C-terminus. The domain was predicted from amino-acid 279 to 342 with less significance than the required threshold. This result suggests that XoBphP probably has different signal transduction mechanisms for its intracellular function.     

Functional analysis of <Emphasis Type="Italic">Xa3/Xa26</Emphasis> family members in rice resistance to <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Plant disease resistant (R) genes are frequently clustered in the genome. The diversity of members in a complex R-gene family may provide variation in resistance specificity. Rice Xa3/Xa26, conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo) encodes a leucine-rich repeat (LRR) receptor kinase-type protein and belongs to a multigene family, consisting of Xa3/Xa26, MRKa, MRKc and MRKd in rice cultivar Minghui 63. MRKa and MRKc are intact genes, while MRKd is a pseudogene. Complementary analyses showed that MRKa and MRKc could not mediate resistance to Xoo when regulated by their native promoters, but MRKa not MRKc conferred partial resistance to Xoo when regulated by a strong constitutive promoter. Plants carrying truncated XA3/XA26, which lacked the kinase domain, were compromised in their resistance to Xoo. However, the kinase domain of MRKa could partially restore the function of the truncated XA3/XA26 in resistance. MRKa and MRKc showed similar expression pattern as Xa3/Xa26, which expressed only in the vascular systems of different tissues. The expressional characteristic of MRKa and MRKc perfectly fits the function of genes conferring resistance to Xoo, a vascular pathogen. These results suggest that although MRKa and MRKc cannot mediate bacterial blight resistance nowadays, they may be once effective genes for Xoo resistance. Their expressional characteristic and sequence similarity to Xa3/Xa26 will provide templates for generating novel recognition specificity to face the evolution of Xoo. In addition, both LRR and kinase domains encoded by Xa3/Xa26 and MRKa are the functional determinants and MRKa-mediated resistance is dosage-dependent. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional analysis of <Emphasis Type="Italic">pilQ</Emphasis> gene in <Emphasis Type="Italic">Xanthomanas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>, bacterial blight pathogen of rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease in rice. A virulence-attenuated mutant strain HNU89K9 of X. oryzae pv. oryzae (KACC10331), with a transposon insertion in the pilQ gene was used for this study. The pilQ was involved in the gene cluster pilMNOPQ of the Xoo genome. Growth rate of the pilQ mutant was similar to that of wild-type. At level of amino acids, PilQ of Xoo showed that a high sequence identities more than 94% and 70% to Xanthomonas species and to Xyllela fastidiosa, respectively but a low sequence homology less than 30% to other bacterial species. The twitching motility forming a marginal fringe on PSA media was observed on colony of the wild-type strain KACC10331, but not in mutant HNU89K9. Wild-type Xoo cells formed a biofilm on the surface of the PVC plastic test tube, while the mutant strain HNU89K9 did not form a biofilm. The results suggest that the pilQ gene of X. oryzae pv. oryzae plays a critical role in pathogenicity, twitching motility, and biofilm formation.     

Effect of modified glucose catabolism on xanthan production in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In this study, the glucose 6-phosphate dehydrogenase gene (XOO2314) was inactivated in order to modulate the intracellular glucose 6-phosphate, and its effects on xanthan production in a wild-type strain of Xanthomonas oryzae were evaluated. The intracellular glucose 6-phosphate was increased from 17.6 to 99.4 μmol g⁻¹ (dry cell weight) in the gene-disrupted mutant strain. The concomitant increase in the glucose 6-phosphate was accompanied by an increase in xanthan production of up to 2.23 g l⁻¹ (culture medium). However, in defined medium supplemented with 0.4% glucose, the growth rate of the mutant strain was reduced to 52.9% of the wild-type level. Subsequently, when a family B ATP-dependent phosphofructokinase from Escherichia coli was overexpressed in the mutant strain, the growth rate was increased to 142.9%, whereas the yields of xanthan per mole of glucose remained approximately the same.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Detection of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in seeds using a specific TaqMan probe

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.     

Zinc Uptake Regulator (<Emphasis Type="Italic">zur</Emphasis>) Gene Involved in Zinc Homeostasis and Virulence of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in Rice

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. In order to understand the gene of zinc uptake regulator (zur) involved in virulence of the pathogen in rice, we generated a mutant OSZRM by homologous suicide plasmid integration. The mutant failed to grow in NYGB medium supplemented with Zn²⁺ or Fe³⁺ at a concentration of 500 μM or 6 mM, whereas the wild-type strain grew well at the same conditions. The zur mutant was hypersensitive to hydrogen peroxide and exhibited reduction catalase activity and the production of extracellular polysaccharide (EPS). Interestingly, the mutant showed a reduction in virulence on rice but still kept triggering hypersensitive response (HR) in tobacco. When the mutant was complemented with the zur gene, the response was recovered to wild-type. These results suggested that zur gene is a functional member of the Zur regulator family that controls zinc and iron homeostasis, oxidative stress, and EPS production, which is necessary for virulence in X. oryzae pv. oryzae. Wanfeng Yang and Yan Liu contributed equally to this work     

Isolation and characterization of rice mutants compromised in<Emphasis Type="Italic"> Xa21</Emphasis>-mediated resistance to<Emphasis Type="Italic"> X. oryzae</Emphasis> pv.<Emphasis Type="Italic"> oryzae</Emphasis>

The rice gene, Xa21, confers resistance to diverse races of Xanthomonas oryzae pv. oryzae (Xoo) and encodes a receptor-like kinase with leucine-rich repeats in the extra-cellular domain. To identify genes essential for the function of the Xa21 gene, 4,500 IRBB21 (Xa21 isogenic line in IR24 background) mutants, induced by diepoxybutane and fast neutrons, were screened against Philippine race six (PR6) Xoo for a change from resistance to susceptibility. From two greenhouse screens, 23 mutants were identified that had changed from resistant to fully (6) or partially (17) susceptible to PR6. All fully susceptible mutants carried rearrangements at the Xa21 locus as detected by PCR and Southern hybridization. For the partially susceptible mutants, no changes were detected at the Xa21 locus based on Southern and PCR analyses. However, two of these mutants were confirmed via genetic analysis to have mutations at the Xa21 locus. Partially susceptible mutants exhibited variation in level of susceptibility to different Xoo strains, suggesting that they may carry different mutations required for the Xa21-mediated resistance. The mutants identified in this study provide useful materials for dissecting the Xa21-mediated resistance pathway in rice.Communicated by D.J. Mackill     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Two coiled-coil regions of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> harpin differ in oligomerization and hypersensitive response induction

Hpa1_Xoo (harpin) is a type III secreted protein of the rice blight bacterial pathogen Xanthomonas oryzae pv. oryzae that elicits a hypersensitive response (HR) in nonhost tobacco. Hpa1_Xoo is predicted to contain two potential coiled-coil (CC) regions, one at the N-terminus with a high probability of formation, and one at the C-terminus with a lower probability of formation. We constructed several CC-equivalent peptides by a chemosynthetic method, and investigated the structure–function of the predicted Hpa1_Xoo CC regions, using biophysical and biochemical approaches. Both peptides elicited an HR in tobacco. Mutant versions of the N- and C-terminal peptides that were predicted to disrupt or favor CC formation were generated. The resulting altered HR activity and oligomerization indicated that the N-terminal CC region is essential for eliciting HR, but the C-terminus is not. The results also indicate that a 14-residue fragment (LDQLLCQLISALLQ) within the N-terminal CC region is a minimal and independent functional element for HR-induction in tobacco leaves. We propose that HR-induction requires a specific oligomerization of the CC regions of Hpa1_Xoo.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.