A high-throughput method for early screening of coffee (<Emphasis Type="Italic">Coffea</Emphasis> spp.) genotypes for resistance to root-knot nematodes (<Emphasis Type="Italic">Meloidogyne</Emphasis> spp.)

Root-knot nematodes (Meloidogyne spp.) threaten the livelihood of millions of farmers producing coffee worldwide. The use of resistant plants either as cultivars or rootstocks appears to be the single most effective method of control. A screening method was developed to evaluate large populations of plants for resistance to root-knot nematodes. Two coffee cultivars, one susceptible and the other resistant to Meloidogyne paranaensis, were grown under controlled conditions in two substrates: a commercial sieved potting compost and an inert substrate containing sand with a water-absorbent synthetic polymer. Plant growth and development and nematode multiplication were compared for two inoculation dates (2 and 8 weeks after planting) and two evaluation dates (eight and 13 weeks after inoculation). Root growth, but not nematode multiplication, was influenced by the choice of substrate. Evaluation of the differences in root weight and nematode numbers between the different cultivars, substrates and dates of inoculation suggested that an optimal condition could be defined. The best discrimination between susceptible and resistant plants was found in the experiment where inoculation occurred at 2 weeks after planting and evaluation occurred at 8 weeks after inoculation. Because the total duration of this experiment was only 3 months, high-throughput evaluation was possible, opening up new possibilities for screening large germplasm collections and studying the genetic control of root-knot nematode resistance in coffee.     

<Emphasis Type="Italic">In vitro</Emphasis> bacterization of banana (<Emphasis Type="Italic">Musa</Emphasis> spp.) with native endophytic and rhizospheric bacterial isolates: Novel ways to combat <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Compared to conventional planting material, micropropagated plantlets are highly susceptible to Fusarium wilt because they are free from beneficial root inhabitants. We aimed to introduce mixtures of beneficial microbes in the plantlets in the rooting medium under in vitro conditions rather than by field applications. Endophytes and rhizobacteria from different banana cultivars and plantation areas were screened and characterized. Under in vitro conditions, banana tissue culture plantlets were bacterized with the prospective endophytes, Bacillus subtilis strain EPB56 and EPB10 and the rhizobacteria, Pseudomonas fluorescens strain Pf1 and effects of in vitro bacterization were investigated against Fusarium oxysporum f. sp. cubense race 1 under glasshouse and field conditions. Inoculation of bananas during micropropagation allowed for the omission of minerals and salts as well as vitamins from the growing media while resulting in plantlets close to double size compared to the controls with full strength media. All endophyte and rhizobacteria strains tested resulted in significant reductions in Fusarium infection in the glasshouse and field and in significantly better plant growth. The three-way combination of bacteria resulted in 78% disease reduction and more than doubled the yields compared to the untreated controls across two field experiments. Three-way inoculation led to yields of 23 and 24 kg/ bunch compared to chemical disease control (13; 15 kg/bunch) and untreated controls (10; 13 kg/bunch) in the two field experiments. Under glasshouse conditions, activity of defence enzymes was significantly increased by all inoculation treatments. Inoculation in vitro led to the establishment of the microorganisms in the plant system before delivering to the farming community. Micropropagation combined with the establishment of a beneficial microbial consortium should complement the micropropagated plants for easier adaptation under field conditions.     

Identification and characterization of <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler causing <Emphasis Type="Italic">Ceratonia</Emphasis> Blight disease of carob (<Emphasis Type="Italic">Ceratonia siliqua</Emphasis> L.) in Turkey

A new dagger nematode, Xiphinema tica n. sp., is described and illustrated from several populations extracted from soil associated with several crops and wild plants in Costa Rica. The new dagger nematode is characterised by a moderate body size (3276–4240 μm), a rounded lip region, ca 13.5 μm wide, separated from body contour by a shallow depression, amphidial fovea large, stirrup-shaped, a moderately long odontostyle ca 135 μm long, stylet guiding ring located at ca 122 μm from anterior end, vulva almost equatorial (50–54%), well-developed Z-organ, with heavy muscularised wall containing in the most of specimens observed two moderately refractive inclusions variable in shape (from round to star-shaped), with uterine spines and crystalloid bodies; female tail short, dorsally convex-conoid, with rounded end and a small peg, with a c’ ratio ca 0.8, bearing two or three pairs of caudal pores and male absent. The unique and novel uterine differentiation based on the coexistence of a well-developed Z-organ mixed with uterine spines and crystalloid bodies in Xiphinema prompted us to update and include this combination of characters in the polytomous key of Loof and Luc (1990). Integrative diagnosis was completed with molecular data obtained, using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, partial 18S–rDNA and the partial mitochondrial gene cytochrome c oxidase subunit 1 (coxI). The phylogenetic relationships of this species with other Xiphinema spp. indicated that X. tica n. sp. was monophyletic to the other species from the morphospecies Group 4, Xiphinema oleae.     

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.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

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.     

Natural enemies of <Emphasis Type="Italic">Diaspis echinocacti</Emphasis> in Greece and first records of <Emphasis Type="Italic">Aphytis debachi</Emphasis> and <Emphasis Type="Italic">Plagiomerus diaspidis</Emphasis>

Two hymenopteran parasitoids of the cactus scale Diaspis echinocacti (Bouché) (Hemiptera: Diaspididae) on Opuntia ficus-indica (L.) Mill. (Cactaceae) are recorded in Greece. Aphytis debachi Azim, 1963 (Aphelinidae) is first recorded for Europe and Plagiomerus diaspidis Crawford, 1910 (Encyrtidae) is first recorded for Greece. Preliminary data on phenology and natural enemies of the scale D. echinocacti on O. ficus-indica are presented. Parasitism of D. echinocacti by P. diaspidis reached 86% in southern Greece (Kalamata) and parasitism by A. debachi reached 9.3% and 12% in Kalamata and Athens, respectively. Two predators, Cybocephalus fodori Endrödy-Youga (Coleoptera: Nitidulidae) and a mite species (Prostigmata: Bdellidae), were found to be associated with D. echinocacti.     

Infection process of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> in oats (<Emphasis Type="Italic">Avena sativa</Emphasis> L.)

Fusarium head blight in small grain cereals has emerged as a major problem in the Nordic countries. However, the impact of this disease in oats has been less investigated than in other cereals. For this reason we have studied the infection process (the optimal time of infection and infection pathways) of Fusarium graminearum in oats and its subsequent effects on kernel infection, deoxynivalenol (DON) content and germination capacity. In a field experiment the oat cultivar Morton was spray-inoculated at different developmental stages, and the highest kernel infection and DON content and lowest germination percentage were observed when inoculation took place at anthesis. Field grown oats affected by a natural Fusarium head blight epidemic and spray-inoculated field and greenhouse oats were used to study the infection pathway. Results showed that the fungus entered primarily through the floret apex into the floret cavity, where it could infect via the internal surfaces of the palea, lemma and caryopsis. Both visual symptoms and fungal infections started at the apical portions of the florets and progressed to the basal portions. Hyphae of F. graminearum grew more profusely on the anthers than on other floret parts during initial stages of infection. Disease development within the oat panicle was slow and is primarily by physical contact between adjoining florets, indicating that the long pedicels give Type II resistance in oats.     

Distribution of <Emphasis Type="Italic">Dickeya</Emphasis> spp. and <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> in naturally infected seed potatoes

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

Potential of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. and <Emphasis Type="Italic">Talaromyces flavus</Emphasis> for biological control of potato stem rot caused by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Sixteen isolates belonging to 11 species of Trichoderma (T. asperellum, T. ceramicum, T. andinensis, T. orientalis, T. atroviride, T. viridescens, T. brevicompactum, T. harzianum, T. virens, T. koningii and T. koningiopsis) were evaluated for biological control of potato (Solanum tuberosum) stem rot caused by Sclerotinia sclerotiorum. In dual culture tests, all antagonists significantly reduced sclerotia formation, and were able to inhibit radial growth of the pathogen. Growth inhibition by production of volatile and non-volatile inhibitors was also measured in in vitro tests. In screening the most efficient species of Trichoderma, establishment of mycelium on sclerotia and sclerotia lysis were also considered as important biocontrol qualities. Excluding T. asperellum, T. brevicompactum, T. andinensis and T. harzianum, all tested Trichoderma species were able to lyse sclerotia. The sclerotia-destroying species of Trichoderma and one isolate of Talaromyces flavus were tested in greenhouse tests and during 2 years of field experimentation during the 2007 and 2008 cropping seasons. After one aerial application of spore suspension in greenhouse trials, T. koningii, T. virens, T. ceramicum and T. viridescens were the most effective bio-agents and reduced significantly disease severity, and the least biocontrol efficacy was observed in T. flavus. Under field conditions and after five soil and foliar applications of spore suspension, all tested antagonists reduced significantly disease incidence. T. viridescens followed by T. ceramicum showed the best results. T. flavus and T. orientalis were less effective than other tested antagonists in both field trials.     

Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout Japan.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

Functional analysis of the melanin biosynthesis genes <Emphasis Type="Italic">ALM1</Emphasis> and <Emphasis Type="Italic">BRM2</Emphasis>-<Emphasis Type="Italic">1</Emphasis> in the tomato pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.     

Infection process of <Emphasis Type="Italic">Gilbertella persicaria</Emphasis> in papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.) fruits

Gilbertella persicaria is a pathogenic fungus recently reported as a causative agent of soft rot in papaya fruits. Here the interactions between G. persicaria and papaya fruits was analyzed under laboratory conditions using histological techniques and optical microscopy to elucidate the process of pathogenesis. Healthy and disinfested fruits of papaya cv. Maradol were also inoculated with a suspension of sporangiospores of G. persicaria. Tissue sections were cut, which were subjected to differential staining with safranin-fast green for different times. Sporangiospores presumably adhered to the cuticle of the fruit by 3 h post inoculation (hpi) and germinated by 6 hpi; invasive intracellular hyphae were growing in host cells by 9 hpi. By 15 hpi, fruit epidermis was macerated, presumably by enzymatic activity reported for mucoral fungal species and appeared as a wet-looking lesion on the cuticle. Fruit mesocarp was colonized by 30 hpi, and asexual reproduction structures had formed by 48 hpi. This process of infection and disease development of G. persicaria in papaya fruits corresponds to that used by pathogens with a necrotrophic lifestyle.     

Identity and pathogenicity of <Emphasis Type="Italic">Fusarium</Emphasis> species associated with crown rot on wheat (<Emphasis Type="Italic">Triticum</Emphasis> spp.) in Turkey

An extensive survey was carried out to collect Fusarium species colonizing the lower stems (crowns) of bread wheat (Triticum aestivum L.) and durum wheat (T. durum Desf.) from different wheat growing regions of Turkey in summer 2013. Samples were collected from 200 fields representing the major wheat cultivation areas in Turkey, and fungi were isolated from symptomatic crowns. The isolates were identified to species level by sequencing the translation elongation factor 1-alpha (TEF1-α) gene region using primers ef1 and ef2. A total of 339 isolates representing 17 Fusarium species were isolated. The isolates were identified as F. culmorum, F. pseudograminearum, F. graminearum, F. equiseti, F. acuminatum, F. brachygibbosum, F. hostae, F. redolens, F. avenaceum, F. oxysporum, F. torulosum, F. proliferatum, F. flocciferum, F. solani, F. incarnatum, F. tricinctum and F. reticulatum. Fusarium equiseti was the most commonly isolated species, accounting for 36% of the total Fusarium species isolated. Among the damaging species, F. culmorum was the predominant species being isolated from 13.6% of sites surveyed while F. pseudograminearum and F. graminearum were isolated only from 1% and 0.5% of surveyed sites, respectively. Six out of the 17 Fusarium species tested for pathogenicity caused crown rot with different levels of severity. Fusarium culmorum, F. pseudograminearum and F. graminearum caused severe crown rot disease on durum wheat. Fusarium avenaceum and F. hostae were weakly to moderately virulent. Fusarium redolens was weakly virulent. However, F. oxysporum, F. equiseti, F. solani, F. incarnatum, F. reticulatum, F. flocciferum, F. tricinctum, F. brachygibbosum, F. torulosum, F. acuminatum and F. proliferatum were non-pathogenic. The result of this study reveal the existence of a wide range of Fusarium species associated with crown rot of wheat in Turkey.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

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.