Multiple copies of <Emphasis Type="Italic">AMT2</Emphasis> are prerequisite for the apple pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis> to produce enough AM-toxin for expressing pathogenicity

The apple pathotype of Alternaria alternata produces the cyclic depsipeptide AM-toxin and causes Alternaria blotch of apple. Previously, we cloned AMT2 from the apple pathotype as an orthologue of AFTS1, which is required for biosynthesis of the decatrienoic acid ester AF-toxin I of the strawberry pathotype. These genes were predicted to encode aldo-keto reductases involved in biosynthesis of a common precursor, 2-hydroxy-isovaleric acid, of AF-toxin I and AM-toxin. In this study, we analyzed the function of AMT2 in AM-toxin biosynthesis in the apple pathotype. DNA gel blot analysis of the apple pathotype strain IFO8984 with five restriction enzymes suggested that this strain has a single copy of AMT2 in the genome. However, gene disruption experiments showed that IFO8984 probably has three copies of AMT2. We made mutants having one or two copies of AMT2 disrupted. The single-copy mutants produced less AM-toxin than did the wild type and were still as pathogenic as the wild type. The two-copy mutants produced trace or undetectable amounts of AM-toxin and were markedly reduced in pathogenicity. Thus, AMT2 was verified to be required for AM-toxin biosynthesis and hence pathogenicity. The fact that the two-copy mutants have a remaining copy of AMT2 suggests that multiple copies of AMT2 are prerequisite for the pathogen to produce enough AM-toxin for full pathogenicity.     

Cloning of the pathogenicity-related gene <Emphasis Type="Italic">FPD1</Emphasis> in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB110097     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

<Emphasis Type="Italic">Alternaria alternata</Emphasis> apple pathotype (<Emphasis Type="Italic">A. mali</Emphasis>) causes black spot of European pear

A disease caused by Alternaria alternata occurred on the leaves of European pear cultivar Le Lectier in Niigata Prefecture, Japan, and was named black spot of European pear. In conidial inoculation tests, the causal pathogen induced not only small black lesions on the leaves of European pear cultivar Le Lectier, but severe lesions on the leaves of apple cultivar Red Gold, which is susceptible to the A. alternata apple pathotype (previously called A. mali) causing Alternaria blotch of apple. Interestingly, the apple pathotype isolate showed the same pathogenicity as the European pear pathogen. HPLC analysis of the culture filtrates revealed that A. alternata causing black spot of European pear produced AM-toxin I, known as a host-specific toxin of the A. alternata apple pathotype. AM-toxin I induced veinal necrosis on leaves of Le Lectier and General Leclerc cultivars, both susceptible to the European pear pathogen, at 5?×?10^?7 M and 10^?6 M respectively, but did not affect leaves of resistant cultivars at 10^?4 M. PCR analysis with primers that specifically amplify the AM-toxin synthetase gene detected the product of expected size in the pathogen. These results indicate that A. alternata causing black spot of European pear is identical to that causing Alternaria blotch of apple. This is the first report of European pear disease caused by the A. alternata apple pathotype. This study provides a multiplex PCR protocol, which could serve as a useful tool, for the epidemiological survey of these two diseases in European pear and apple orchards.     

Cytological events in tomato leaves inoculated with conidia of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f. sp. <Emphasis Type="Italic">hordei</Emphasis> and <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01

Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.     

Specific inhibition of spore germination of <Emphasis Type="Italic">Alternaria brassicicola</Emphasis> by fistupyrone from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TP-A0569

Fistupyrone (FP), a metabolite from Streptomyces sp. TP-A0569, inhibited the in vivo infection of Chinese cabbage seedlings by Alternaria brassicicola. To detect the possible action sites of FP, the effect of FP on the infection behavior of A. brassicicola and A. alternata was investigated. When spores of A. brassicicola were suspended in FP solution and inoculated on host leaves, FP at 0.1ppm significantly inhibited spore germination, appressorial formation, and infection hypha formation of A. brassicicola. Host-specific AB-toxin production and lesion formation by A. brassicicola spores were also reduced significantly by treatment with FP 1ppm. The effect of FP seemed to be irreversible because significant washing of FP-treated spores with distilled water (DW) did not change the inhibitory effects. In contrast, A. alternata isolates such as Japanese pear pathotype, apple pathotype, and saprophyte behaved almost equally in both FP- and DW-treated spores. Mycelial dry weight in potato dextrose broth and mycelial diameters on potato dextrose agar, gelatin glucose agar, and Czapek solution agar of both A. brassicicola and A. alternata were not different with or without addition of FP. These results indicate that FP at low concentrations has a fungicidal effect on spores of A. brassicicola but not on spores of A. alternata; FP also does not affect the vegetative phase of these fungi.     

Mutational analysis of type III effector genes from <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>

Xanthomonas citri subsp. citri, the causal agent of citrus canker, relies extensively on a type III secretion system for infection by delivering type III effectors into host cells. In the genus Xanthomonas, two major regulators, HrpG and HrpX, are involved in the expression of genes encoding the type III secretion system. Twenty-three candidate type III effectors were identified as targets for analysis. The involvement in pathogenicity of 20 candidate effector genes in X. citri strain 306 (Xcc-306) was investigated using site-directed mutagenesis. Pathogenicity assays in grapefruit of 19 genes using site-directed mutagenesis revealed that none of the mutants demonstrated to have reduced ability to cause canker disease. A mutation in the TAL effector pthA4 ^– resulted in loss of hypertrophy although no changes were observed in bacterial growth in leaves. Mutations in hrpG, hrpX, or hrpA genes displayed a complete loss of pathogenicity. Moreover, all mutants maintained the ability to trigger a hypersensitive response (HR) in non-host tomato. In contrast to previous studies, hrpG ^– , hrpX ^– and hrpA ^– mutants also retained the ability to elicit an HR in tomato, indicating the presence of an Hrp independent elicitor in Xcc-306.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Comparison of the development <Emphasis Type="Italic">in planta</Emphasis> of a pyrrolnitrin-resistant mutant of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and its sensitive wild-type parent isolate

Botrytis cinerea is able to build-up resistance to pyrrolnitrin, an antibiotic produced by diverse biocontrol agents, possibly compromising the durability of this method of disease control. The development of two near-isogenic lines of B. cinerea differing in their level of resistance to pyrrolnitrin was compared in tomato plants and on PDA medium. In tomato plants, significant differences in the percentage of infected petioles 1 day after inoculation and in symptom progression on petioles and stems were observed between the resistant mutant and the sensitive wild-type parent, suggesting a difference in their level of aggressiveness. Cytohistological investigations revealed that conidia of both near-isogenic lines germinated 6 h after inoculation and mycelium developed within petiole tissues 12 h after inoculation. However, while the wild-type parent isolate spread throughout the petiole and rapidly invaded the stem tissues via the leaf-abscission zone 72 h after inoculation, the pyrrolnitrin-resistant mutant failed to extend beyond petiole tissues to invade the stem. Moreover, 72 h after inoculation, the mycelial development of the pyrrolnitrin-resistant mutant was accompanied by abnormal glycogen accumulation and chlamydospore-like cell formation. In contrast, wild-type parent mycelium was normally structured with intensive colonization of stem tissues. Additionally, on PDA medium the mycelium of the pyrrolnitrin-resistant mutant was less vigorous than the wild-type isolate. These results suggest that the acquisition of pyrrolnitrin-resistance in B. cinerea is accompanied by changes in mycelial structure and reduction in mycelial growth, leading to a noticeable loss of aggressiveness on tomato plants.     

Cloning and characteristics of <Emphasis Type="Italic">Brn1</Emphasis> gene in <Emphasis Type="Italic">Curvularia lunata</Emphasis> causing leaf spot in maize

The full length cDNA of the Brn1 was first cloned, and then expression of the Brn1 was analyzed and the function was identified by silencing technology. Results show that the full length cDNA of the C. lunata Brn1 gene contains 1001 base pairs and an 801 bp open reading frame encoding 267 amino acids. Semi-quantitative PCR analysis shows that the expression of Brn1 at 96 h is significantly higher than at 24 and 72 h (p < 0.05) in both the highly virulent isolate CX-3 and the weakly virulent isolate DD60. Brn1-silenced transformants were light brown in culture filtrate, and have significantly reduced toxin production relative to the wild-type. These results imply that Brn1 gene in C. lunata is not only involved in 1,8-dihydroxynaphthalene melanin synthesis, but is also relatively associated with toxin biosynthesis of the pathogen.     

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.     

Infectivity of a Japanese isolate of <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01 to a European tomato cultivar resistant to <Emphasis Type="Italic">O. lycopersici</Emphasis>

The infectivity of a Japanese isolate of tomato powdery mildew, Oidium neolycopersici KTP-01, to tomato cultivars was examined using a resistant cultivar Grace bred in The Netherlands to O. lycopersici, which was recently proposed to be renamed O. neolycopersici. Grace was severely infected with KTP-01, and its susceptibility was similar to that on susceptible tomato cultivars Moneymaker and Ponderosa, suggesting that KTP-01 differs in pathogenicity on tomatoes from those of European and American isolates.     

First report of leaf spot caused by <Emphasis Type="Italic">Alternaria alternata</Emphasis> on <Emphasis Type="Italic">Drimia maritima</Emphasis>

Drimia maritima (squill) is a historically important medicinal plant. During the spring of 2016, small, yellow leaf spots, which became brown and finally necrotic, were observed on squill plants in Kohgiluyeh and Boyer-Ahmad Provinces in Iran. A fungus was consistently isolated from infected leaves and identified as Alternaria alternata based on morphological and phylogenetic analyses. Pathogenicity tests confirmed A. alternata to be the causal agent of the newly observed leaf spot disease. This is the first report of leaf spot on D. maritima caused by A. alternata in the world.     

Relation between pathogenicity and genetic variation within <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A–D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.     

Visualisation of <Emphasis Type="Italic">hrp</Emphasis> gene expression in <Emphasis Type="Italic">Xanthomonas euvesicatoria</Emphasis> in the tomato phyllosphere

The plasmid pUFZ75 conferred constitutive GFP expression on the bacterial pathogen Xanthomonas euvesicatoria (syn. X. campestris pv. vesicatoria). Colonisation of the tomato phyllosphere and invasion of tomato leaves by X. euvesicatoria was examined using both fluorescence and confocal laser scanning microscopy. Xanthomonas euvesicatoria established a limited population on the tomato leaf surface, primarily occupying the depressions between epidermal cells and around the stomata, prior to invasion of the leaf via the stomata and subsequent growth within the substomatal chamber and the leaf apoplast. Additionally, hrp-gfp fusions were used to report on the temporal and spatial expression of hrp genes during epiphytic colonisation and invasion. Xanthomonas euvesicatoria cells carrying hrpG- and hrpX-gfp reporter constructs were not fluorescent in vitro on non-hrp-inducing LB agar but did exhibit a low level of fluorescence on the leaf surface within 24 h of inoculation, particularly in the vicinity of stomata. Cells carrying hrpG- and hrpX-gfp fusions exhibited high levels of fluorescence 72 h after inoculation in the substomatal chamber and the leaf apoplast. Cells carrying the hrpF-gfp fusion were slightly fluorescent on LB agar and showed no further increase in fluorescence on the leaf surface by 24 h after inoculation, but did show a significant increase in fluorescence 72 h after inoculation in the substomatal chamber and apoplast. The apparent low-level induction of the regulators hrpG and hrpX on the tomato leaf surface may suggest that some of the genes of the X. euvesicatoria HrpG/HrpX regulon are up- or down-regulated prior to invasion of the stomata while still on the leaf surface.     

<Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>, a potential biocontrol agent on apple rust mite <Emphasis Type="Italic">Aculus,schlechtendali</Emphasis> and interactions with some fungicides <Emphasis Type="Italic">in vitro</Emphasis>

The apple rust mite Aculus schlechtendali (Nal.) (Acari: Eriophyidae), is a main pest in apple-growing areas in Ankara, Turkey, and chemical control applications have some limitations. Entomopathogenic fungi have a potential for biological control of mites. In this study, an entomopathogenic fungus, Paecilomyces lilacinus (Thom) Samson (Deuteromycota: Hyphomycetes), was first isolated from the mite cadavers on Japanese crab apple leaves and pathogenicity of the fungus was observed in different inoculum densities and relative humidities. The pathogen caused up to 98.22% mortality of the mite population. The effects of some fungicides on the entomopathogenic fungus were determined in in vitro studies. Carbendazim, penconazole and tebuconazole were the most effective fungicides on mycelial growth of P. lilacinus, with EC₅₀ values under 3 μg ml⁻¹. In spore germination tests, captan, mancozeb, propineb were the most effective fungicides, followed by tebuconazole, penconazole, nuarimol and chlorothalonil. Sulphur could not inhibit the conidia germination totally at 5,000 μg ml⁻¹. Copper oxychloride and fosetyl-al prevented conidia formation at concentrations above 1,000 μg ml⁻¹.     

Effect of Phenylpyrrole-resistance Mutations on Ecological Fitness of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and their Genetical Basis in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Mutants of Botrytis cinerea and Ustilago maydis highly resistant to fludioxonil were isolated at a high frequency, after nitrosoguanidine or UV mutagenesis, respectively, and selection on media containing fludioxonil. Tests on the response of mutant strains to high osmotic pressure resulted in the identification of two fludioxonil-resistant phenotypes (FLD_osm/s and FLD_osm/r), regarding the sensitivity to high osmolarity. Approximately 95% of fludioxonil-resistant mutants were found to be more sensitive to high osmotic pressure than the wild-type parent strains. Genetic analysis of phenylpyrrole-resistance in the phytopathogenic basidiomycete U. maydis, showed that fludioxonil-resistance was coded by three unlinked chromosomal loci (U/fld-1, U/fld-2 and U/fld-3), from which only the U/fld-1 mutation coded an osmotic sensitivity similar to that of the wild-types. Cross-resistance studies with fungicides from other chemical groups showed that the mutations for resistance to phenylpyrroles affect the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides, but not to the benzimidazoles, anilinopyrimidines, phenylpyridinamines, hydroxyanilides or the sterol biosynthesis inhibiting fungicides. A study of fitness parameters in the wild-type and fludioxonil-resistant mutants of B. cinerea, showed that all osmotic sensitive (B/FLD_osm/s) isolates had significant reductions in the characteristics determining saprophytic fitness such as mycelial growth, sporulation, conidial germination and sclerotial production. Contrary to that, with the exception of mycelial growth, the fitness parameters were unaffected or only slightly affected in most of the osmotic resistant (B/FLD_osm/r) isolates. Tests on cucumber seedlings showed that the osmotic-sensitive strains were significantly less pathogenic compared with the wild-type and B/FLD_osm/r strains of B. cinerea. Preventative applications of the commercial products Saphire 50 WP (fludioxonil) and Rovral 50 WP (iprodione) were effective against lesion development on cotyledons by the wild-type and the mutant strains of B. cinerea that were resistant to the anilinopyrimidine cyprodinil (B/CPL-27) and to the hydroxyanilide fenhexamid (B/FNH-21), but ineffective, even at high concentrations, against disease caused by the fludioxonil-resistant isolates (B/FLD) and a mutant strain resistant to the dicarboximide iprodione (B/IPR-1). Experiments on the stability of the fludioxonil-resistant phenotype showed a reduction of resistance, mainly in osmotic-sensitive isolates, when the mutants were grown on inhibitor-free medium. A rapid recovery of the high resistance was observed after mutants were returned to the selection medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain of B. cinerea, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of osmotic-resistant mutants was higher than the osmotic-sensitive ones. Furthermore, competition tests, in planta, showed a significant reduction of the frequency of both phenylpyrrole-resistant phenotypes, with a respective increase in the population of the wild-type strain of the pathogen.     

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.     

The potential use of carvacrol for the control of <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

The protein kinase HOG1 in high osmolarity glycerol pathway is one of mitogen-activated protein kinases and plays an important role in fungi cellular responses to external stress and stimuli. In this study, AlHOG1, a yeast HOG1 homolog, was isolated from tobacco pathogenic fungus Alternaria longipes and its disrupted mutants were generated using homologous recombination. Compared with the wild-type strain, the mutants grew slower under Minimal-Media, produced less conidia on Potato-Dextrose-Agar and showed a retardation phenotype in conidial germination rate. Colony color observation indicated that AlHOG1 most likely negatively regulated melanin production under nutrients-deficient or osmotic stress. The same action mode of AlHOG1 on hyphal growth in response to certain carbon or nitrogen sources was speculated. Additionally, the disrupted strains were sensitive to osmotic and oxidative stress, and showed increased resistance to dicarboximide, phenylpyrrole and carbendazim fungicides. Meanwhile, results from the comparative analysis between the AlHOG1- and the AlHK1 (encoding a group III histidine kinase)-disrupted mutants showed that AlHOG1 mediated-pathway was partly regulated by the upstream AlHK1 kinase for cellular resistance to salts osmotic stress and that AlHK1 was a primary regulator for cellular sugar and fungicides stress adaptation and likely modulated both AlHOG1-mediated pathway and other unknown mechanisms. Noteworthily, AlHOG1 but not AlHK1 was confirmed to be associated with high concentration glycerol adaptation, which is the first report to our knowledge. Overall, our results indicated that AlHOG1 was involved in multiple physiological processes in A. longipes, including mycelial and conidial development, melanin production, nutrients sensing and multi-stress responses.