Small molecules cause virulence attenuation of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. oryzae,the pathogen causing bacterial blight of rice

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight in rice, has developed resistant strains due to rampant usage of chemical pesticides and disease resistant rice cultivars. Virulence attenuating agents could be an alternative strategy to combat this pathogen; therefore, the main objective of the present study was to screen phenolic compounds of plant origin as well as a thiadiazole derivative, acibenzolar S methyl for their effect on various virulence determinants of Xanthomonas oryzae pv. oryzae such as the type three secretion system, motility, extracellular polysaccharide production and secretion of exoenzymes through the type two secretion system. From the compounds screened, cinnamic acid and its derivatives like ferulic acid, caffiec acid and p-coumaric acid showed significant reduction in promoter activity of xopQ, an effector secreted through type three secretion system. None of the compounds used in this study showed substantial inhibitory effect on other virulence determinants indicating that their effect was specific on the type three secretion system except acibenzolar S methyl which, along with inhibitory effects on promoter activity of xopQ, also showed reduction in motility of bacteria. Wheat-bran-derived natural medium was effective for induction of type three and type two secretion systems at detectable levels. We also performed virulence assay that showed significant reduction in disease symptoms confirming cinnamic acid, ferulic acid, caffeic acid, p-coumaric acid as effective virulence attenuating agents of Xanthomonas oryzae pv. oryzae BXO43. These studies demonstrate the potential of selected small molecules for development of new alternative strategies to control the bacterial blight of rice.     

Generation and characterisation of Tn5-tagged <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> mutants that overcome <Emphasis Type="Italic">Xa23</Emphasis>-mediated resistance to bacterial blight of rice

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23. The authors C.-L. Wang, A.-B. Xu contributed equally to this work; Y. Gao and Y.-L. Fan contributed equally to this work.     

Hpa1 secretion via type III secretion system in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In many Gram-negative plant pathogenic bacteria the type III secretion system (TTSS), encoded by hrp genes, is essential for pathogenicity in the host and induction of a hypersensitive reaction (HR) in nonhost plants. The expression of hrp genes has been suggested to be repressed in complex media, whereas it is induced in planta and under certain in vitro conditions. We recently reported that XOM2 medium allows efficient hrp expression by Xanthomonas oryzae pv. oryzae. In this study, we investigated hrp-dependent secretion of proteins by the bacteria in vitro. Using modified XOM2, in which bovine serum albumin was added and the pH was lowered to 6.0, we detected at least 10 secreted proteins and identified one as Hpa1. This is the first evidence of protein secretion via TTSS in X. oryzae pv. oryzae.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

A molecular mechanism of resistance to streptomycin in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzicola</Emphasis>

Xanthomonas oryzae pv. oryzicola, the causal agent of rice leaf streak disease, was found to be sensitive to streptomycin (an aminocyclitol glycoside antibiotic), by inhibition of protein synthesis resulting from interference with translational proofreading. This study aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzicola to streptomycin. Seven streptomycin-resistant mutants were obtained by UV induction or streptomycin selection. These mutants can grow at 100 μg ml⁻¹ of streptomycin while the wild-type strain (RS105) cannot grow at 5 μg ml⁻¹. Sequencing indicated that the rpsL gene encoding ribosomal protein S12 has 375 bp encoding 125 amino acid residues. In all resistant strains, a mutation in which AAG was substituted for AGG (Lys→Arg) occurred either at codon 43 or 88. Two plasmids, pUFRRS and pUFRRX, were constructed by ligating the rpsL gene into the cosmid pUFR034. The plasmids pUFRRS and pUFRRX containing the Lys→Arg mutation of the rpsL gene conferred streptomycin resistance to the sensitive wild-type strain by electroporation. Both transformants, RS1 and RS2, could grow in the medium containing 50 μg ml⁻¹ of streptomycin. A mutation at codon 43 or 88 in rpsL can result in resistance of Xanthomonas oryzae pv. oryzicola to streptomycin.     

Salicylic acid mediated multi-pronged strategy to combat bacterial blight disease (<Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">punicae</Emphasis>) in pomegranate

Pomegranate bacterial blight disease caused by Xanthomonas axonopodis pv. punicae is one of the most serious threat to the pomegranate industry in India. So far, the chemicals used to control this pathogen alone are less effective. In this article, an attempt was made to supplement chemical control through improving the systemic acquired resistance of plant using nutrients such as nitrogen (N) and salicylic acid (SA) as elicitor. The study revealed that foliar application of SA at the rate of 300 ppm increased above-ground dry mass of plant by 64.97%, improved chlorophyll content of leaves and resulted higher concentration of macro-elements and micro-elements, particularly Mn and Zn, in leaves. It also significantly increased the activities of nitrate reductase (NR), superoxide dismutase (SOD), catalase and peroxidase. However, the maximum response of SA was recorded when it was sprayed after soil application of N at 100% of the recommended dose. As a consequence, this combination recorded the least bacterial blight infection with average severity of 11.58% followed by without-N application under challenge inoculation of the pathogen. However, sustainable production cannot be realized without N application. The integration of this strategy for activation of defence mechanism of plant with the bactericide streptocycline resulted in further reduction of bacterial blight disease incidence and severity under field condition to very low levels (3.84% and 18.57% respectively).     

Population features of <Emphasis Type="Italic">Xanthomonas arboricola</Emphasis> pv. <Emphasis Type="Italic">pruni</Emphasis> from <Emphasis Type="Italic">Prunus spp.</Emphasis> orchards in northern Italy

Bacterial leaf/fruit spot and canker of stone fruits, caused by Xanthomonas arboricola pv. pruni, is a recurrent disease in Italy. A set of 23 strains has been isolated in peach and plum orchards in an intensively stone fruit cultivated area located in north-eastern Italy. They were all identified as X. arboricola pv. pruni by means of phytopathological and serological features: hypersensitive reaction on bean pods, pathogenicity test on immature peach or plum fruitlets, identification by immunofluorescence assay and conventional PCR. Phylogenetic analysis based on sequencing of the gyrB housekeeping gene of the isolates showed that they formed a unique clade, well characterised and separated from other xanthomonads. An insight into the genetic population features was attempted by rep-PCR analysis, using the ERIC, REP and BOX primers. The combined rep-PCR fingerprints showed a slight intra-pathovar variation within our isolates, which grouped in five close clusters. Copper resistance has been assessed in vitro for our whole X. arboricola pv. pruni collection, highlighting that two isolates show a level of resistance in vitro up to 200 ppm of copper. Nonetheless, the copLAB gene cluster, present in many other species of Xanthomonads, was not detected in any isolate, confirming the presence of a still unknown mechanism of copper detoxification in our Xanthomonads arboricola pv. pruni tolerant/resistant strains.     

Detection of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">manihotis</Emphasis>, the causal agent of cassava bacterial blight diseases in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) in Ghana by polymerase chain reaction

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of cassava bacterial blight (CBB) disease. CBB is a major constraint to cassava cultivation in Ghana. In this study, a survey was conducted in eight regions of Ghana to assess the presence of CBB disease. Out of the eight regions visited, CBB, though at different prevalence, was observed in five regions. Cassava plants samples showing suspected bacterial blight symptoms were collected for analysis by Polymerase Chain Reaction (PCR). The results of the analysis showed that Ashanti region had the highest prevalence in percentage of CBB, which recorded (70%), followed by Volta region (60%); Brong Ahafo region (40%); Eastern region (40%) and Greater Accra region (20%). Morphological examination of the putative pathogen was carried out on Cefazolin trehalose agar (CTA) and Nutrient agar (NA) media. The isolates were subjected to conventional PCR using Xanthomonas genus specific primer, RST2/RST3, Xam specific Variable Number Tandem Repeat (VNTRs) loci, XaG1_67F/R and X-gumD primers, which produced 840, 446 and 402 bp, respectively. The isolates also tested positive with SYBR Green fluorescent dye, using Real-time PCR. The resulting PCR products were sequenced and analyzed using a BLASTn program, which revealed homology between 93 and 100% with several other Xam strains retrieved from GenBank nucleotide database. The pathogenicity test of the isolates on the susceptible Esam cassava variety produced symptoms typical of Xam and the pathogen was consistently re-isolated from the inoculated cassava plants and thereby satisfying the Koch’s postulates.     

Identification of a highly virulent strain of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">malvacearum</Emphasis>

A highly virulent strain (HVS) of Xanthomonas axonopodis pv. malvacearum (Xam) was first reported in Africa in 1983, infecting all commercial cultivars of cotton including the immune cv. ‘101–102B’. The HVS was considered to be a new race of pathovar malvacearum (race 20). Here we studied a HVS (GSPB 2388) isolated in Sudan, which causes symptoms that clearly differed from the typical angular water-soaked spots of bacterial bright of cotton. Our investigations showed that extracellular cellulase activity of this HVS was higher than that of the control strain GSPB 1386 (race 18). Additionally, SDS-PAGE indicated that the HVS cell wall contained short LPS molecules with fewer O-chain repeating units, lacking in GSPB 1386. The higher cellulase activity and the distinct lipopolysaccharide of HVS are correlated with the higher virulence and deviating symptom formation. Rep-PCR fingerprinting showed that the HVS was very closely related to other strains of Xam.     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

Population structure of <Emphasis Type="Italic">Eleusine</Emphasis> isolates of <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> and its evolutionary implications

Population structure of Eleusine isolates of Pyricularia oryzae (Magnaporthe oryzae) was examined using DNA markers. On the basis of rDNA sequences, Eleusine isolates were divided into two groups. One group clustered with Triticum isolates, while the other clustered with Eragrostis isolates. This grouping was supported by DNA fingerprinting with three repetitive elements: MGR586, MGR583, and grasshopper. These results suggest that the population of Eleusine isolates is composed of at least two groups that evolved independently from the original population of P. oryzae. Most of the isolates that were collected just after an outbreak of finger millet blast in the 1970s had almost identical fingerprint profiles although they were collected in distant prefectures. This result supports the idea that the outbreak was caused by seed transmission of a particular strain of Eleusine isolates.     

Antifungal activity of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. culture filtrates against the rice pathogen <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

An ethyl acetate extract of a culture filtrate (ECF) from an unidentified fungal isolate O821 was evaluated for antifungal activity against the rice pathogen Magnaporthe oryzae. The O821-ECF significantly inhibited spore germination, appressorium formation, and mycelial growth of M. oryzae, and its antifungal activity was heat-stable. It also significantly suppressed the number and size of blast lesions. In an analysis of the ITS sequence of this isolate, it shared similarities with species of the fungus Biscogniauxia. These results suggest that isolate O821 of the genus Biscogniauxia produces a heat-stable antifungal compound(s) in its culture filtrate.     

Production and preliminary characterization of monoclonal antibodies raised against <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">mangiferaeindicae</Emphasis>

Bacterial black spot disease of mango is caused by Xanthomonas campestris pv. mangiferaeindicae (Xcm), which consists of two genotypically and phenotypically distinct groups of strains. Monoclonal antibodies (MABs) were produced – 15 against CFBP 1717, a group I strain, and 9 against CFBP 2919 (yellow-pigmented), a group II strain – and were analyzed for their characteristics. On the avidin-biotin peroxidase complex enzyme-linked immunosorbent assay, the dilution limit of the MABs was between 100 and 200000 and was 10 times higher when measured on the corresponding ascitic fluid. All kinds of isotypes were represented among the MABs. All the Japanese Xcm strains, designated group I by hrp-restriction fragment length polymorphism (RFLP) analysis, reacted equally with MAB 1A7H12G3, which is the most specific for all but one worldwide group I strains, and to only one strain among group II. Also, to various extents, serological heterogeneity inside the two groups was consistently differentiated based on isozyme and RFLP analyses. MAB 1E2E1 against CFBP2919, because of its narrow specificity, and MAB 1A7H12G3 against CFBP1717, because of its broad specificity, will be useful for epidemiological studies or general control of the pathogen.     

Discrimination of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> races among strains from northwestern Spain by <Emphasis Type="Italic">Brassica</Emphasis> spp. genotypes and rep-PCR

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a severe seedborne disease of Brassica crops around the world. Nine races are recognized, being races 1 and 4 the most aggressive and widespread. The identification of Xcc races affecting Brassica crops in a target area is necessary to establish adequate control measures and breeding strategies. The objectives of this study were to isolate and identify Xcc strains from northwestern Spain by using semi-selective medium and pathogenicity tests, determine the existing races of Xcc in this area by differential series of Brassica spp., and evaluate the use of repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) to differentiate among the nine existing Xcc races. Seventy five isolates recovered from infected fields were identified as Xcc. Race-typing tests determined the presence of the following seven pathogen races: 1, 4, 5, 6, 7, 8 and 9. Race 4 was the most frequent in Brassica oleracea and race 6 in Brassica rapa crops, therefore breeding should be focussed in obtaining resistant varieties to both races. Cluster analysis derived from the combined fingerprints showed four groups, but no clear relationship to race, crop or geographical origin was found. Rep-PCR analysis was found not to be a reliable method to discriminate among Xcc races, therefore race typing of Xcc isolates should be done by using the differential series of Brassica spp. genotypes or another alternative approach.