Influence of rice-isolated bacteria on chitinase production by the biocontrol bacterium Serratia marcescens strain B2 and the genetically modified rice epiphytic bacterium

Chitinolytic activity of the biocontrol bacterium Serratia marcescens strain B2 was inhibited by bacteria isolated from rice, even though its growth was not affected. Antifungal activity of the strain against Pyricularia oryzae was also reduced under the influence of these bacteria. In contrast, the rice-epiphytic bacterium Erwinia ananas NR1, transformed with chitinase gene chiA derived from strain B2, had high chitinolytic activity regardless of the presence of the bacteria isolated from rice. Therefore, the introduction of an antagonistic factor gene with a promoter from the recipient into the epiphytic bacteria may prove useful in the development of effective biocontrol agents.     

Induced Resistance to Rice Blast by Antagonistic Bacterium, <Emphasis Type="Italic">Serratia marcescens </Emphasis>Strain B2

An antagonistic bacterium, Serratia marcescens strain B2, controlled rice blast after being sprayed onto rice phylloplane, as did the bacterial suspension when poured into rhizosphere soil of rice plants. Three days after root treatment, rice blast conidia were sprayed onto rice foliage. A week after pathogen inoculation, rice blast was suppressed and lesions caused by the pathogen decreased in size. Brown deposits were observed around sites of pathogen infection after root treatment. Induced resistance was not associated with an increase in the activitiy of peroxidase, phenylalanine ammonia lyase, tyrosine ammonia lyase, β-1,3-glucanase, β-1,4-glycosidase, N-acetylhexosaminidase or chitinase. However, lipoxygenase levels were elevated after the root treatment with strain B2 following inoculation with the pathogen. Strain B2 was not detected in rice foliage after root treatment. These data suggest that strain B2 induced resistance against rice blast caused by Pyricularia oryzae. Received 1 November 2001/ Accepted in revised form 25 January 2002     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.     

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.     

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.     

Cloning of various promoters for foreign gene expression in Erwinia ananas

We constructed a promoter-trap plasmid, pEGFP-V1, to isolate various promoters for foreign gene expression in the leaf-colonizing bacterium Erwinia ananas NR-1. A library was constructed in pEGFP-V1 by introducing genomic DNA fragments upstream of the promoterless EGFP gene to transform E. ananas cells. The library, which consisted of 3500 E. ananas transformants was screened for GFP expression. We found nine strong GFP-expressing clones from the library. Furthermore, we characterized the clones by restriction analysis, sequencing, primer extension analysis, and then quantification of promoter activity. Selected promoters, specifically two (PCF9 and PCF53), gave strong gene expression in E. ananas. Our results indicate that pEGFP-V1 is a useful tool for screening DNA fragments with strong promoter activity in E. ananas.     

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.     

Races of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in Australia and genes with resistance to these races revealed through host resistance screening in monogenic lines of <Emphasis Type="Italic">Oryza sativa</Emphasis>

Rice blast is the most serious disease threat to rice production worldwide. It is difficult to control due to the complex diversity and wide geographic distribution of the causal pathogen Magnaporthe oryzae. In Australia, rice blast occurs in northern Australia but remains exotic to the main south-eastern rice growing area; however, there is the potential for rice blast to threaten this area; in addition, rice production is currently expanding from south-eastern Australia into northern Australia, which makes rice blast a major concern and challenge to rice industry in Australia. Prior to this study, there was lack of information on the race status of M. oryzae present in Australia and on how to manage the disease through host resistance. The races of rice blast isolates collected in northern Australia was characterised based on the disease reactions of eight standard rice differentials used in an international race differential system. The following studies revealed genes conferring resistance to these races through investigating the responses of 25 monogenic rice lines with targeted resistance gene against different races. The rice blast isolates were characterised into five races: IA-1, IA-3, IA-63, IB-3 and IB-59. Genes Pi40, Piz-t, Pi9, Pi5(t) and Pi12(t) exhibited resistance to all the isolates belonging to five races. In addition, two genes showed complete resistance to multiple races, viz. Pi9 that showed complete resistance to races IA-1, IA-3, IA-63 and IB-3 and Pita2 that had complete resistance to races IA-3, IB-3 and IB-59. This study provides information about the races of M. oryzae in Australia. Genes identified conferring resistance to multiple races will not only streamline the identification via molecular markers of imported rice varieties with resistance to rice blast in Australia, but will also allow the Australian rice breeding program to develop new varieties with broad-spectrum resistance to rice blast and pyramid multi-gene resistance into Australian rice varieties.     

Antifungal activities of hyoscyamine and scopolamine against two major rice pathogens: <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> and <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The antifungal activities of hyoscyamine and scopolamine, major alkaloids extracted from the desert plant Hyoscyamus muticus, against two rice pathogens, Magnaporthe oryzae and Rhizoctonia solani, were studied. The minimum inhibitory concentration of hyoscyamine that resulted in distinctive inhibition (MIC₅₀) was 1 μg/ml for both fungi. Exposure to hyoscyamine caused the leakage of electrolytes from the mycelia of both fungi. Hyoscyamine (>1 μg/ml) irreversibly delayed or inhibited conidial germination and appressorium formation in M. oryzae grown on polystyrene plates. Hyoscyamine effectively inhibited the attachment of conidia to the surface of rice (Oryza sativa) leaves and inhibited appressorium formation on the leaves. A high concentration of scopolamine (1000 μg/ml) also delayed or inhibited conidial germination in M. oryzae, but conidial germination was restored after washing the conidia with water. Antifungal activity of hyoscyamine was reduced by scopolamine. Magnaporthe oryzae infection was significantly suppressed (by >95%) in leaves of intact rice plants treated with hyoscyamine (10 μg/ml). Moreover, 10 μg hyoscyamine/ml significantly reduced the disease severity index for sheath blight to ≤0.2, when compared with the disease index of control plants (>7.0). Hyoscyamine (>20 μg/ml) completely inhibited sclerotial germination and development of R. solani by delaying the initiation, maturation, and melanization of the sclerotia. These results suggest that tropane alkaloids may be useful for controlling blast and sheath blight diseases of rice and for studying the mechanisms that regulate conidial germination in M. oryzae and sclerotial germination and development in R. solani.     

Overwintering of <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> in wild infected foxtails

The rice blast fungus Pyricularia oryzae mainly overwinters in infested rice organs stored indoors, whereas it is difficult or impossible for the pathogen to overwinter outdoors. By contrast, blast pathogens infecting weed grasses must overwinter outdoors every winter to continue their life cycle. In this study, we investigated the overwintering location of P. oryzae infecting wild, green, and giant foxtails to identify the mechanism that enables them to overwinter. Recovery of P. oryzae was tested in seeds of wild foxtail collected from the soil surface from December to April over three winters. No P. oryzae was recovered from the seed samples of any wild foxtail collected at the ends of the three experimental periods in April. Recovery was also tested from blast lesions on leaves and seeds sampled from withered green foxtail in the experimental field of Saga University from November to April during two winters. In contrast to seeds on the soil surface, P. oryzae survived in lesions and seeds at the ends of the two experimental periods during April, suggesting that withered host plants could be the overwintering site of the pathogen. Rice plants are reaped and removed from paddy fields after harvesting. Thus, withered, standing plants may be available solely to blast pathogens infecting wild grasses, possibly explaining the higher winter survival frequency of weed pathogens than that of rice blast pathogens outdoors.     

Green-odour compounds have antifungal activity against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Four green-odour compounds—trans-2-hexenal, cis-3-hexenol, n-hexanal, and cis-3-hexenal—were applied (0.85 μg ml⁻¹ as vapour) to rice plants in laboratory conditions to observe their biological activity against the phytopathogenic fungus Maganporthe oryzae, which causes rice blast disease worldwide. Two compounds, trans-2-hexenal and cis-3-hexenal, showed remarkable disease suppression efficacy (99.7% and 100% suppression, respectively), while n-hexanal had moderate (86.5%) and cis-3-hexenol had weak (20.8%) disease-suppressing effects. Pre-application and post-application of trans-2-hexenal or cis-3-hexenal had slight effects on blast incidence, suggesting that these compounds had direct effects to suppress M. oryzae infection. In fact, trans-2-hexenal and cis-3-hexenal exhibited a growth suppression effect on M. oryzae. Interestingly, these two compounds inhibited appressorium formation at lower concentrations than the growth suppression. Studies on the hypersensitive response (HR)-like reaction and plant β-1,3-glucanase activity in rice plant confirmed that induced resistance was not the major factor involved in the disease suppression mechanism. Results of this study conclusively showed that trans-2-hexenal and cis-3-hexenal possess potent inhibitory activities against the growth and the appressorium formation of M. oryzae and could be used as antifungal agents to significantly reduce M. oryzae infections in rice.     

Comparative colonisation by virulent versus avirulent <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> on wild <Emphasis Type="Italic">Oryza australiensis</Emphasis>

Pyricularia oryzae (rice blast) conidial development at pre-penetration stage determines success or otherwise of infection inside the rice host plants. Studies on conidial germination and growth on the leaf surface in commercial rice (Oryza sativa) report differently, dependent upon host type and level of blast resistance. Although wild rice (O. australiensis) is known to be an alternative host of blast, the interaction between P. oryzae conidia and wild O. australiensis on its leaf surface has not been previously studied. We found significant (P?<?0.001) differences in conidial development between two blast isolates with different virulence in terms of conidial germination, germ tube growth and appressoria formation on both wild and cultivated rice. Conidial germination at 6 h post-inoculation (hpi) for the virulent isolate was significantly (P?<?0.001) delayed. Germ tubes of the avirulent isolate conidia grew significantly (P?<?0.001) faster and with significantly (P?<?0.001) longer germ tubes than from virulent conidia. Appressoria development for the virulent isolate was significantly (P?<?0.001) faster at its later growth stages of 12 and 18 hpi when approximately 100% of germ tubes formed appressoria. In contrast, formation rate of appressoria for the avirulent isolate was significantly (P?<?0.001) slower and only reached 76% of germ tubes forming appressoria. Appressoria formation on O. australiensis was significantly (P?<?0.001) greater than the formation on O. sativa for both virulent and avirulent P. oryzae at 12 hpi, a clear indication that host type influences the extent of appressoria formation.     

First report on <Emphasis Type="Italic">Enterobacter</Emphasis> sp. causing soft rot of <Emphasis Type="Italic">Amorphophallus konjac</Emphasis> in China

In summer 2010, stems and corms of Amorphophallus konjac with soft rot were collected in Hubei Province, China. Plants were inoculated with the isolated bacterium and developed the same symptoms, and the reisolated pathogen was identified as Enterobacter sp., based on morphology, pathogenicity and 16S rDNA sequence analysis.     

<Emphasis Type="Italic">Bchex</Emphasis> virulence gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis>: characterization and functional analysis

We previously identified Bchex as a highly expressed gene during filamentous growth in Botrytis cinerea. The gene encodes the principal protein of the Woronin body and has been shown to seal septal pores in response to cellular damage. In the present study, Southern blot analysis of genomic DNA indicated that the gene exists as a single copy in the B. cinerea genome. The gene was differentially expressed during various developmental stages: expression was high in germinating conidia and the mycelial stage and lower in resting conidia and the appressorial stage. For functional analyses, homologous recombination was used to obtain a ΔBchex knockout mutant. Growth of the mutant was strongly reduced growth in complete medium and in defined media with sucrose, fructose or pectin as the carbon source. After detached tomato leaves were inoculated with the Bchex mutant, lesion development was markedly reduced compared to the control, suggesting that Bchex participates in normal growth, germination and virulence of this fungus.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.     

Real-time Scorpion-PCR detection and quantification of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> on pear leaves and flowers

A specific primer couple (E3–E4) amplifying a single DNA fragment of 111 bp from plasmid pEA29 was designed to identify, detect and quantify Erwinia amylovora by real-time Scorpion-PCR. Specificity of primers and probe was assessed both by means of BLAST analyses and by using genomic DNA from a large number of E. amylovora isolates and other bacteria. In Scorpion-PCR, the limit of detection was of 1 pg of total DNA and a high correlation (r = 0.999) was achieved between target DNA quantity and cycle threshold (Ct). Combining two sequential amplifications with conventional reported primers (PEANT1–PEANT2) and Scorpion primers (E3 Scorpion-E4) the detection limit was of 1 fg (nested Scorpion-PCR). Using serial dilution of the bacterial suspensions the limit of detection was 3.2 × 10⁴ CFU ml⁻¹ in Scorpion-PCR and 2.8 × 10² CFU ml⁻¹ in nested Scorpion-PCR. Real-time PCR combined with effective procedures for DNA extraction enabled the detection and the quantification of the epiphytic population of E. amylovora in the washings of flowers and leaves of artificially inoculated pear. A significant correlation (r = 0.92) was achieved between pathogen CFU on semi-selective media and the corresponding target DNA concentration evaluated by real-time PCR.