Diversity of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.     

Genetic mapping with dispersed repeated sequences in the rice blast fungus: mapping the SMO locus

Summary The SMO genetic locus in strains of the fungus Magnaporthe grisea that infect weeping lovegrass, directs the formation of correct cell shapes in asexual spores, infection structures, and asci. We have identified and characterized a Smo^– strain of M. grisea that infects rice. The smo mutation in this strain segregates as a single gene mutation and is allelic to previously identified smo alleles. A marked reduction in pathogenicity co-segregates with the Smo^– phenotype, suggesting that the SMO locus plays a role in rice pathogenicity. A family of dispersed repeated DNA sequences, called MGR, have been discovered in the nuclear DNA of M. grisea rice pathogens. Genetic crosses between Smo^– rice pathogens and Smo⁺ non-rice pathogens were used to follow the segregation of the SMO locus and individual MGR sequences. Using DNA blot analysis with cloned MGR hybridization probes, we mapped the SMO locus to a chromosomal region flanked by two closely linked MGR sequences. We demonstrated that the copy number of MGR sequences could be reduced in subsequent crosses to non-rice pathogens of M. grisea, and that new MGR sequences did not occur following meiosis indicating that these sequences are stable in the genome. We conclude that restriction fragment polymorphism mapping with cloned MGR sequences as hybridization probes is an effective way to map genes in the rice blast fungus.     

Population structure and dynamics of Magnaporthe grisea in the Indian Himalayas.

The population genetics of Magnaporthe grisea, the rice blast pathogen, were analyzed in a center of rice diversity (the Uttar Pradesh hills of the Indian Himalayas) using multilocus and single-, or low-copy, DNA markers. Based on DNA fingerprinting with the multilocus probe MGR586 and single-locus probes, 157 haplotypes clustered into 56 lineages (at >/=70% MGR586 band similarity, each with unique single-locus profiles) and high diversity indices were detected among 458 isolates collected from 29 sites during 1992-1995. Most valleys sampled had distinct populations (73% of the lineages were site specific) with some containing one or a few lineages, confirming the importance of clonal propagation, and others were very diverse. Widely distributed lineages suggested that migration occurs across the region and into the Indo-Gangetic plains. Repeated sampling at one site, Matli, (170 isolates, 1992-1995) yielded 19 lineages and diversity significantly greater than that reported from similar samples from Colombia and the Philippines. Analysis of allelic associations using pairwise comparisons and multilocus variance analysis failed to reject the hypothesis of gametic phase equilibrium. The Matli population shifted from highly diverse in 1992 to almost complete dominance by one lineage in 1995. Such population dynamics are consistent with recombination followed by differential survival of clonal descendants of recombinant progeny. At another site, Ranichauri, population (n = 84) composition changed from 2 to 11 lineages over 2 yr and yielded additional evidence for equilibrium. Sexually fertile and hermaphrodite isolates of both mating types were recovered from rice in both Matli and Ranichauri. We demonstrate that Himalayan M. grisea populations are diverse and dynamic and conclude that the structure of some populations may be affected to some extent by sexual recombination.     

Synthetic oligonucleotide probes for detection of mercury-resistance genes in environmental freshwater microbial communities in response to pollutants

Mercury-resistance genes were detected byin situ hybridization using new synthetic oligonucleotide probes specific formerA andmerB genes according to the published sequences of the corresponding enzymes. These DNA probes were used for the detection of specific mercury-resistant microorganisms isolated from the Rhine River which had been polluted 3 years previously in 1986. Mercuric reductase and organomercurial lyase genes persist in the bacterial genome even after the disappearance of the pollutant but are absent in axenic amoebae. A total of 49 bacterial isolates showed DNA homologies with the³²P-labelled DNA probes and 15 free-living amoebae were selected due to their harboured symbiotic mercury-resistant bacteria.     

Relationship between Phylogeny and Pathotype for the Bacterial Blight Pathogen of Rice

Several transposable elements were isolated from the genome of Xanthomonas oryzae pv. oryzae. These elements and an avirulence gene isolated from X. oryzae pv. oryzae were used as hybridization probes for a collection of X. oryzae pv. oryzae strains from the Philippines. Each of the sequences was present in multiple copies in all strains examined and showed distinct patterns of hybridizing bands. Phenograms were derived from the restriction fragment length polymorphism data obtained for each of the individual probes and for pooled data from multiple probes. The phenograms derived from the different probes differed in topology and, on the basis of bootstrap analysis, were not equally robust. For all of the probes, including the avirulence gene, some groups (even some haplotypes) consisted of multiple races. The strains were grouped into four major clusters on the basis of the two probes giving the highest bootstrap values. These groups were inferred to represent phylogenetic lineages. Three of the six races of X. oryzae pv. oryzae appeared in more than one of the lineages, and another was present in two sublineages. For three of the races, strains representing different phenetic groups were inoculated on rice cultivars carrying 10 resistance genes. Two new races were differentiated, corresponding to pathogen lineages identified by DNA typing. On the basis of DNA and pathotypic analyses, together with information on the spatial and temporal distribution of the pathogen types from this and other studies, a general picture of X. oryzae pv. oryzae evolution in the Philippines is presented.     

Origins of host-specific populations of the blast pathogen Magnaporthe oryzae in crop domestication with subsequent expansion of pandemic clones on rice and weeds of rice

Rice, as a widely and intensively cultivated crop, should be a target for parasite host shifts and a source for shifts to co-occurring weeds. Magnaporthe oryzae, of the M. grisea species complex, is the most important fungal pathogen of rice, with a high degree of host specificity. On the basis of 10 loci from six of its seven linkage groups, 37 multilocus haplotypes among 497 isolates of M. oryzae from rice and other grasses were identified. Phylogenetic relationships among isolates from rice (Oryza sativa), millet (Setaria spp.), cutgrass (Leersia hexandra), and torpedo grass (Panicum repens) were predominantly tree like, consistent with a lack of recombination, but from other hosts were reticulate, consistent with recombination. The single origin of rice-infecting M. oryzae followed a host shift from a Setaria millet and was closely followed by additional shifts to weeds of rice, cutgrass, and torpedo grass. Two independent estimators of divergence time indicate that these host shifts predate the Green Revolution and could be associated with rice domestication. The rice-infecting lineage is characterized by high copy number of the transposable element MGR586 (Pot3) and, except in two haplotypes, by a loss of AVR-Co39. Both mating types have been retained in ancestral, well-distributed rice-infecting haplotypes 10 (mainly temperate) and 14 (mainly tropical), but only one mating type was recovered from several derived, geographically restricted haplotypes. There is evidence of a common origin of both ACE1 virulence genotypes in haplotype 14. Host-haplotype association is evidenced by low pathogenicity on hosts associated with other haplotypes.     

Biological control of rice bacterial blight by Lysobacter antibioticus strain 13-1

Bacterial leaf blight (BB) is a worldwide destructive rice disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo). A novel strain of Lysobacter antibioticus, which was isolated from the rhizosphere of rice in Yunnan Province of China, can significantly inhibit the growth of various phytopathogenic bacteria and fungi, especially BB pathogen Xoo. In greenhouse experiments, whole bacterial broth culture (WBC) of strain 13-1 was more effective in reducing BB than other components of the culture, with disease suppression efficiency up to 69.7%. However, bacterial cells re-suspended in water, cell-free culture extracts, and heated cultures also significantly reduced BB severity. Suppression efficiencies ranged from 79.0% to 61.8% for undiluted to 100-fold dilution treatments and from 57.6% to 31.7% when the WBC of strain 13-1 (10⁸ CFU/mL) was applied at 3 days and 7 days prior to pathogen inoculation, respectively. In three field trials, strain 13-1 reduced BB incidence by 73.5%, 78.3%, and 59.1%, respectively. Disease suppression by strain 13-1 varied significantly among different rice cultivars, although efficacy was not directly related to the susceptibility level of the cultivars. Efficacy of biocontrol was also affected by different pathogen isolates, with some isolates of Xoo being more sensitive to 13-1 suppression than others. These results suggest that antibiotics and density of colonization on leaves may be involved for biological control of rice BB by strain 13-1. To our knowledge, this is the first report of L. antibioticus being a potential biocontrol agent for rice bacterial blight.     

中国稻瘟病菌的致病性和其DNA指纹图谱间关系的研究

通过在稻瘟病菌Pyricularia oryza。基因组文库中的筛选,找到一个散布的并具有基因组特异性的层重复顺序POR6。本文报道用这一株针对6个日本菌株和26个中国北方菌株进行DNA指纹作图的结果。其中22个中国北方菌株按其杂交带型百分相似率被分成8个株系。一些在我们实验室保存的菌株用传统方法鉴定发现在转管过程中会发生致病性变异。当用POR6作探针与这些菌株NNA的EcoRV酶切片段杂交时,检测出它们的无性世代中出现数条EcoRV多态性片段。     

Common genotypes (RFLP) within a diverse collection of yellow-green aspergilli used to produce traditional Oriental fermented foods

 DNA fingerprinting was performed on 72 strains of Aspergillus oryzae and 9 strains of Aspergillus sojae isolated from chu (China) or koji (Japan) mold inoculum used in the production of traditional Oriental fermented beverages or foods including soy sauce, miso, and sake. The cultures were deposited with the ARS Culture Collection (NRRL) between 1909 and 2001. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. All strains of A. sojae that we examined produced an identical DNA fingerprint and belong to the same DNA fingerprint group (GTAo-9). Strains of A. oryzae were distributed among 41 DNA fingerprint groups, including GTAo-12 represented by 11 strains, GTAo-19 represented by 5 strains, GTAo-5 and GTAo-15 each represented by 4 strains, and GTAo-8, GTAo-17, and GTAo-24 each represented by 3 strains. Thirty-three single strain isolates of A. oryzae produced unique fingerprints. Our data offer evidence suggesting that (1) the successful domestication of A. parasiticus genotypes yielding A. sojae occurred far less frequently than among genotypes of A. flavus var. oryzae; and (2) some Aspergillus genotypes employed in different fermentations and regions were derived from a common ancestral clonal population. Received: February 18, 2002 / Accepted: April 22, 2002     

DNA fingerprinting of Indian isolates of Xanthomonas oryzae pv. oryzae

 A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)₁₀, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)₁₀ and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity. Received: 8 December 1996 / Accepted: 20 December 1996     

DNA addition or deletion is associated with a major karyotype polymorphism in the fungal phytopathogen Colletotrichum gloeosporioides

Summary A 1.2 Mb minichromosome resolved by pulsed-field electrophoresis was present in two independent race 3 isolates of Colletotrichum gloeosporioides causing Type B anthracnose specifically on Stylosanthes guianensis cv. Graham in Australia. This chromosome was absent in duplicate isolates representing races 1, 2 and 4 which infect other S. guianensis cultivars. A gene library was prepared specifically from the 1.2 Mb mini-chromosome and ten independent DNA clones unique to this chromosome were identified by differential hybridisation to whole chromosome probes. All of the ten selected probes hybridised only to the 1.2 Mb minichromosome unique to the race 3 isolates but not to any chromosome in isolates of the other races. These ten probes also hybridised only to restriction-digested DNA of race 3 and were thus both chromosome- and strain-specific for Type B C. gloeosporioides. Hybridisation analysis of NotI fragments of the 1.2 Mb minichromosome with these sequences indicated that they were not tightly clustered on the chromosome. These data demonstrate that the variation in the occurrence of the 1.2 Mb minichromosome did not arise by rearrangement of the genome of a progenitor strain but involved either large scale deletion or addition of DNA. The 1.2 Mb minichromosome did not contain a cloned high-copy-number repeat sequence present on all other mini- and maxichromosomes, suggesting addition from a genetically distinct strain. All ten chromosome-specific DNA probes hybridised to a 2.0 Mb chromosome in all races of C. gloeosporioides causing Type A anthracnose on Stylosanthes spp. including S. guianensis. Restriction fragment length polymorphism analysis demonstrated that only 15% of the hybridising restriction fragments of the Type A 2.0 Mb chromosome and the 1.2 Mb Type B race 3 minichromosome were identical. This indicated that it is unlikely that the 1.2 Mb minichromosome of the race 3 Type B pathogen was recently introgressed from-the Type A pathogen.     

Effect of Temperature and Illumination on Growth and Reproduction of the Green Alga Ulva fenestrata

The combined effect of temperature (5, 10, 15, and 20°C) and illumination (40 and 60 mE/(m² s)) on growth and reproduction of the green marine alga Ulva fenestrata P. et R. from the sublittoral zone of Amursky Bay, Sea of Japan, was studied in the laboratory environment in the months April–July, 2000. It was demonstrated that the temperature of 5°C and illumination of 40 mE/(m² s) are the most favorable for maintaining the vegetative mass of the algae. A water temperature of 10°C and illumination of 40 mE/(m² s) are the optimum conditions for vegetative growth of U. fenestrata thalli. A temperature decrease and increase by 5°C reduces the growth rate on average by 30%. Sporo- and gametogenesis in U. fenestrata are the most regular (every 10 days) and occupy the greatest disk area at a water temperature of 15°C and illumination of 40 mE/(m² s). Vegetative growth of thalli is sharply inhibited at the stage of cell preparation to gametogenesis a day before the beginning of gamete formation.     

Relationship between growth rate of Helminthosporium oryzae isolates on calluses of rice cultivars and their disease reaction on rice plants

Rice cultivars showed differential reaction to various isolates of Helminthosporium oryzae, the brown spot pathogen. The calluses obtained from those cultivars behaved in a similar manner to the mycelial growth of pathogenic isolates on them. However, amount of inoculum, size of the callus and period of incubation influenced the reaction of the callus to the fungal isolates.     

Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers

Four genes of rice,Oryza sativa L., conditioning resistance to the bacterial blight pathogenXanthomonas oryzae pv.oryzae (X. o. pv.oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed betweenxa-5 and RFLP marker lociRZ390, RG556 orRG207 on chromosome 5.Xa-3 andXa-4 were linked to RFLP locusXNpb181 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker toXa-10, also located on chromosome 11, was the RAPD locusO07 ₂₀₀₀ at a distance of 5.3 cM. From this study, the conventional map [19, 28] and two RFLP linkage maps of chromosome 11 [14, 26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes,Xa-4 +xa-5 andXa-4 +Xa-10. Lines carryingXa-4 +xa-5 andXa-4 +Xa-10 were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs of genes were resistant to more of the isolates than their single-gene parental lines. Lines carryingXa-4 +xa-5 were more resistant to isolates of race 4 than were either of the parental lines (quantitative complementation). No such effects were seen forXa-4 +Xa-10. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.     

Reduced but accurate translation from a mutant AUA initiation codon in the mitochondrial COX2 mRNA of Saccharomyces cerevisiae

The gene encoding the XorII methyltransferase (M · XorII) was cloned from Xanthomonas oryzae pv. oryzae and characterized in Escherichia coli. The M · XorII activity was localized to a 3.1 kb BamHI-BstXI fragment, which contained two open reading frames (ORFs) of 1272 nucleotides (424 amino acids) and 408 nucleotides (136 amino acids). Ten polypeptide domains conserved in other M⁵ cytosine methyltransferases (MTases) were identified in the deduced amino acid sequence of the 1272 ORF. E. coli Mrr⁺ strains were transformed poorly by plasmids containing the XorII MTase gene, indicating the presence of at least one ^MCG in the recognition sequence for M · XorII (CGATCG). The 408 nucleotide ORF was 36% identical at the amino acid level to sequences of the E. coli dcm-vsr gene, which is required for very short patch repair. X. oryzae pv. oryzae genomic DNA that is resistant to digestion by Pvul and XorII hybridizes with a 7.0 kb fragment containing the XorII MTase gene and vsr homolog, whereas DNA from strains that lack M · XorII activity do not hybridize with the fragment.The sequence presented in this paper has been submitted to NCBI; the accession number is U06424     

TheMagnaporthe grisea DNA fingerprinting probe MGR586 contains the 3′ end of an inverted repeat transposon

TheMagnaporthe grisea repeat (MGR) sequence MGR586 has been widely used for population studies of the rice blast fungus, and has enabled classification of the fungal population into hundreds of genetic lineages. While studying the distribution of MGR586 sequences in strains ofM. grisea, we discovered that the plasmid probe pCB586 contains a significant amount of single-copy DNA. To define precisely the boundary of the repetitive DNA in pCB586, this plasmid and four cosmid clones containing MGR586 were sequenced. Only 740 bp of one end of the 2.6-bp insert in the pCB586 plasmid was common to all clones. DNA sequence analysis of cosmid DNA revealed that all the cosmids contained common sequences beyond the cloning site in pCB586, indicating that the repetitive DNA in the fingerprinting clone is part of a larger element. The entire repetitive element was sequenced and found to resemble an inverted repeat transposon. This putative transposon is 1.86 kb in length and has perfect terminal repeats of 42 bp, which themselves contain direct repeats of 16 bp. The internal region of the transposon possesses one open reading frame which shows similarity at the peptide level to the Pot2 transposon fromM. grisea and Fot1 fromFusarium oxysporum. Hybridization studies using the entire element as a probe revealed that some strains ofM. grisea, whose DNA hybridized to the pCB586 probe, entirely lacked MGR586 transposon sequences.     

The Genetic Structure of Populations of Potato virus Y in Japan; Based on the Analysis of 20 Full Genomic Sequences

The genetic structure of Potato virus Y (PVY) populations in Japan was analysed using 20 isolates; five were retrieved from the public DNA sequence databases, and an additional 15 complete genomic sequences were determined using field samples collected in Japan. Recombination and phylogenetic analyses of a total of 149 isolates from Japan and other countries showed that PVY has three major lineages (C, N and O); at least one, two and six sublineages in C, N and O lineages, respectively. One recombination pattern was newly found among Japanese PVY^NTN strain isolates, which was most closely related to the PVY^NTN strain isolates previously found in Europe and North America. On the other hand, PVY^O was a complex of several divergent lineages, and there were at least three non‐recombinant subpopulations in Japan. Studies on nucleotide diversities of populations and phylogenetic relationships of the isolates in the PVY sequences showed that Japanese PVY populations were in part distinct from the European and North American populations.     

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

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