A novel pathosystem to study the interactions between <Emphasis Type="Italic">Lotus japonicus</Emphasis> and <Emphasis Type="Italic">Fusarium solani</Emphasis>

A wilt disease of the model legume Lotus japonicus was observed in a greenhouse in Tokyo, Japan in May 2004. Roots of diseased plants were rotted and dark brown with lesions spreading to lower stems and leaves, resulting in rapid plant death. The causal agent was identified as Fusarium solani based on the morphology. Sequence analysis of rDNA supported the identification. Inoculation of roots of healthy plants with conidia reproduced characteristic disease symptoms, and F. solani was reisolated from lesions, satisfying Koch’s postulates. The isolate also caused chlorotic to necrotic lesions on leaves of healthy plants after wound-inoculation. Infection by F. solani of leaves of L. japonicus was confirmed histologically. Mycelia were observed in the intercellular spaces of parenchymatous tissues in the lesion area and the surrounding tissues. This is the first report of fungal disease on L. japonicus satisfying Koch’s postulates. We named it “Fusarium root rot of L. japonicus” as a new disease. The compatibility of L. japonicus and F. solani is expected to form a novel pathosystem for studying interactions between legumes and fungal pathogens. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB258993 and AB258994.     

Race 3, a new race of<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lactucae </Emphasis>determined by a differential system with commercial cultivars

 Pathogenic variation among 26 Japanese isolates of Fusarium oxysporum f. sp. lactucae (FOL) was tested using 21 lettuce cultivars to select commercial lettuce cultivars as race differential indicators. Cultivar Costa Rica No. 4 was resistant to race 1 but susceptible to race 2, consistent with the conventional standard differential line VP1010. Cultivar Banchu Red Fire was susceptible to race 1 but resistant to race 2, which showed an opposite type of reaction as another differential line VP1013. Cultivar Patriot was susceptible to both races. The resistance reactions of the three cultivars under field conditions were identical with that observed in the seedlings. Thus cv. Costa Rica No. 4 and cv. Banchu Red Fire can be used as differential hosts to identify pathogenic races of FOL. This differential system showed that all FOL isolates obtained from diseased butterhead lettuce in Fukuoka, Japan were new races (i.e., pathogenic to three cultivars). We propose that the new race be designated race 3. Isolates of FOL, the pathogen of Fusarium wilt in lettuce, obtained from California showed the same reaction as that of race 1. Furthermore, the Japanese isolate SB1-1 (race 1) and California isolate HL-2 belonged to the same vegetative compatibility group. Our results suggest that both of the fungi are the same forma specialis. Received: March 25, 2002 / Accepted: August 26, 2002     

Selective media for <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Selective media without pentachloronitrobenzene were developed for quantitative assays of Fusarium oxysporum in soils. Media Fo-G1 and Fo-G2 were effective for naturally infested soils, Fo-W1 and Fo-W2 for wild-type isolates in soils containing a nitrate-nonutilizing (nit) mutant, and Fo-N1 and Fo-N2 for nit mutants. Selective media were made using ammonium citrate dibasic, l-sorbose, econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder for soil dilutions of 100-fold or more (Fo-G1, FoW1 and Fo-N1) and 10-fold (Fo-G2, Fo-W2 and Fo-N2). Potassium chlorate was added to Fo-N1 and Fo-N2. The efficacy for selectively isolating F. oxysporum was confirmed using six soils naturally infested with one of six formae speciales of F. oxysporum and with soil dilutions containing conidia of wild-type strains or nit mutants from the six formae speciales. On Fo-G1 and Fo-G2, most colonies of F. oxysporum were compact and round with purplish or reddish pigment in the reverse. Cylindrocarpon sp. formed colonies as large as those of F. oxysporum but were distinguishable by their colony morphology. Other contaminants such as F. solani, F. moniliforme, and Trichoderma were suppressed by medium ingredients and colonies of F. oxysporum. On Fo-W1 and Fo-W2, colony morphology of F. oxysporum and contaminants corresponded to that on Fo-G1 and Fo-G2, although F. oxysporum failed to produce the pigment. On Fo-N1 and Fo-N2, nit mutants formed clear colonies from 100- and 10-fold soil dilutions, respectively, and contaminants seldom formed large colonies.     

Seed transmission of<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp.<Emphasis Type="Italic">lactucae</Emphasis>

Twenty-seven seed samples belonging to the lettuce cultivars most frequently grown in Lombardy (northwestern Italy), in an area severely affected by Fusarium wilt of lettuce, were assayed for the presence ofFusarium oxysporum on a Fusarium-selective medium. Isolations were carried out on subsamples of seeds (500 to 1500) belonging to the same seed lots used for sowing, and either unwashed or disinfected in 1% sodium hypochloride. The pathogenicity of the isolates ofF. oxysporum obtained was tested in four trials carried out on lettuce cultivars of the butterhead type, very susceptible to Fusarium wilt. Nine of the 27 samples of seeds obtained from commercial seed lots used for sowing in fields affected by Fusarium wilt were contaminated byF. oxysporum. Among the 16 isolates ofF. oxysporum obtained, only one was isolated from disinfected seeds. Three of the isolates were pathogenic on the tested cultivars of lettuce, exhibiting a level of pathogenicity similar to that of the isolates ofF. oxysporum f.sp.lactucae obtained from infected wilted plants in Italy, USA and Taiwan, used as comparison. The results obtained indicate that lettuce seeds are a potential source of inoculum for Fusarium wilt of lettuce. The possibility of isolatingF. oxysporum f.sp.lactucae, although from a low percent of seeds, supports the hypothesis that the rapid spread of Fusarium wilt of lettuce observed recently in Italy is due to the use of infected propagation material. Measures for prevention and control of the disease are discussed. http://www.phytoparasitica.org posting Dec. 16, 2003.     

Genetic diversity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan based on phylogenetic analyses of rDNA-IGS and <Emphasis Type="Italic">MAT1</Emphasis> sequences

Twenty-eight isolates of Fusarium oxysporum f. sp. spinaciae (FOS; the causal agent of spinach wilt) collected from Japan were assessed for mating type and subjected to phylogenetic analysis. Mating type analysis revealed all isolates to be MAT1-2, suggesting that there is no sexual recombination within the population. Phylogenetic analyses based on nucleotide sequences of the ribosomal DNA intergenic spacer (IGS) and the mating type locus (MAT1) suggested that FOS is polyphyletic. The cluster analysis based on IGS showed four phylogenetic groups (S1–S4) among the isolates. Two distinct lineages, S1 and S3, included FOS isolates both of the vegetative compatibility group (VCG) types, 0330 and 0331, demonstrating that VCG differentiation in FOS may not necessarily reflect the phylogenetic relationships based on IGS and MAT1-2-1.     

Characterization of biotin-autotrophic isolates derived from naturally occurring auxotrophic race 2 isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lactucae</Emphasis>

Race 2 isolates of Fusarium oxysporum f. sp. lactucae have been recognized as biotin auxotrophs and consequently have restricted growth on Puhalla's minimal medium (MM), which contains no biotin. Biotin-autotrophic isolates were raised from race 2 isolates through cultural mutation that grew as well on MM as they did on MM supplemented with biotin. These autotrophs were identical to the parental isolates in pathogenicity on race differential cultivars of lettuce (Patriot, Banchu Red Fire, and Costa Rica No. 4), and thus were designated as race 2. A vegetative compatibility test indicated that the autotrophic isolates fell into the same vegetative compatibility group as the parents. Culture filtrates of the autotrophs allowed abundant growth of the parental auxotroph on MM, and, through a competitive enzyme-binding assay, biotin was detected in the culture filtrates. These results suggest that biotin auxotrophy in the natural race 2 isolates has no direct relation to pathogenicity, qualitatively defined as physiological race, or to vegetative compatibility.     

Lack of biocontrol capacity in a non-pathogenic mutant of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis>

The aim of this study was to assess the biocontrol capacity of rev157, a non-pathogenic mutant of a pathogenic strain of Fusarium oxysporum f. sp. melonis (Fom24). Inoculated in association with the virulent parental strain, the mutant rev157 did not protect the host plant (muskmelon) against infection by Fom24. Applied on flax, a non-host plant, the mutant rev157 was not able to protect it against its specific pathogen F. oxysporum f. sp. lini. On the contrary the parental strain Fom24 did protect flax as well as a soil-borne biocontrol strain (Fo47). Since the mutant rev157 was affected neither in its growth in vitro nor in its capacity to penetrate into the roots, it can be speculated that the mutation has affected traits responsible for interactions within the plant. In F. oxysporum the pair of strains Fom24/rev157 is a good candidate to identify genes involved in the biocontrol capacity of F. oxysporum and to test the hypothesis of a link between capacity to induce the disease and capacity to induce resistance in the plant.     

Detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. palmivora</Emphasis> in citrus roots using PCR-RFLP in comparison with other methods

Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.     

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     

Impaired purine biosynthesis affects pathogenicity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis>

The vascular wilt pathogen Fusarium oxysporum f. sp. melonis causes worldwide yield losses of muskmelon. In this study, we characterized a UV-induced non-pathogenic mutant (strain 4/4) of F. oxysporum f. sp. melonis, previously identified as a potential biological control agent. During comparative analysis of vegetative growth parameters using different carbon sources, mutant strain 4/4 showed a delay in development and secretion of extracellular enzymes, compared to the wild type strain. Amendments of the growth medium with yeast extract, adenine or hypoxanthine, but not guanine, complemented the growth defect of strain 4/4, as well as secretion and partial activity of cellulases and endopolygalacturonases, indicating that the strain is an adenine auxotroph. Incubation of strain 4/4 conidia in adenine solution, prior to inoculation of muskmelon plants, partially restored pathogenicity to the mutant strain.     

New race of <Emphasis Type="Italic">Phytophthora vignae</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis>, the causal agent of Phytophthora stem rot of the adzuki bean

 A new race of Phytophthora vignae f. sp. adzukicola, designated race 4, is reported from central and western Hokkaido, Japan. The isolates obtained from diseased plants of a new cultivar, cv. Syumari, which is resistant to races 1, 2, and 3, were determined to be a new race by the pathogenic reaction on a set of differential adzuki bean cultivars (cv. Erimo-shozu, cv. Kotobuki-shozu, cv. Noto-shozu, cv. Urasa-shimane, and cv. Syumari). Received: March 7, 2002 / Accepted: August 13, 2002     

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.     

Virulence and diversity of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f.sp. <Emphasis Type="Italic">hordei</Emphasis> in East China

Four hundred and sixty-one isolates of Blumeria graminis f.sp. hordei were obtained from eight populations occurring on cultivated barley (Hordeum vulgare) at four geographically distant locations in China during 2003 and 2004. Their virulence frequency was determined on 30 differential lines. No isolate was virulent on differential lines possessing the resistance genes Mla1, Mla3, Mla6, Mla7, Mla9, Mla12, Mla13, Mlat, Mlg, Mla10, Mla22, Mla23, Mlp1, Ml(N81) and Mlmw. Virulences to the first nine resistance genes are prevalent in Europe and constitute the main part of genetic distance between Chinese and European populations. Conversely, no isolate was avirulent on the differential lines possessing the genes Mla8 and Ml(Ch). The frequencies of isolates overcoming the genes Mla2, Mla11, Mlk1 and Mlk2 were .4–9.3%, and frequencies of isolates overcoming the genes Mlh, MlLa, Ml(Bw), Mlra, Ml(Ru2), mlw, MlGa, MlWo and Mlnn ranged from 18.2% to 98.7%. Based on reactions of differential lines possessing the genes Mlk1, Mlh, MlLa, Ml(Bw), Mlra and Ml(Ru2), pathotypes were identified and diversity parameters calculated. Eleven of 22 detected pathotypes were found in both years and comprised 94.6% of isolates. Generally, the populations from different locations in 1 year were more closely related than populations collected from the same locations in different years. Complete effectiveness of the resistance genes, for which no corresponding virulences were found, will allow Chinese breeders to access many modern European barley cultivars that are fully resistant to powdery mildew in China, including those possessing the non-host resistance gene mlo.     

Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strains simultaneously colonizing root surfaces

 Green fluorescent protein (GFP)-marked Fusarium oxysporum f. sp. melonis and nonmarked F. oxysporum f. sp. fragariae were stained with neutral red. The neutral red stained vacuoles of the fungi without disturbing GFP fluorescence in the cytoplasm. GFP-marked fungi showed fluorescent hyphae with dark-stained vacuoles, whereas nonmarked fungi were detected as nonfluorescent hyphae with dark-dotted vacuoles. Root colonization by these two fungi was monitored using this method. Microconidia attached similarly to the root surface and elongated vegetative hyphae. Only the pathogenic fungi invaded, causing necrosis at the inoculation site. Thus, the present method enabled us to track simultaneously the various formae speciales of F. oxysporum colonizing the root surface. Received: March 25, 2002 / Accepted: September 27, 2002     

Biological soil disinfestation using ethanol: effect on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis> and soil microorganisms

Better soil disinfestation methods, such as biological soil disinfestation (BSD), that are environmentally safe are increasingly been developed and used because of rising concerns related to environmental risks. We evaluated the efficacy of soil disinfestation using ethanol to control the fungus Fusarium oxysporum f. sp. lycopersici, which causes fusarium wilt of tomato. Survival of bud cells and chlamydospores declined markedly in soil saturated with diluted ethanol solution in the laboratory. In field trials, artificially added nonpathogenic Fusarium oxysporum and indigenous F. oxysporum were both strongly suppressed in soil saturated with 1% ethanol solution; a wheat bran treatment was not as effective. The artificially added fungus was not detected in three of four sites treated with ethanol but was detected in three of four sites amended with wheat bran. Using ethanol in pre-autoclaved soil was not suppressive; thus native microorganisms are essential for the suppression. This ethanol-mediated biological soil disinfestation (Et-BSD) temporarily increased the number of anaerobic bacteria, but the number of fungi and aerobic bacteria was stable. Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) analysis revealed slight but apparent differences in bacterial community structures in the soil treated with Et-BSD compared with the structure in soils after other treatments such as water irrigation and in the control soil, which received neither organic amendment nor irrigation after 15 days. Et-BSD is a potentially effective and easy soil disinfestation method, and its impact on native, beneficial microorganisms is moderate.     

<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> and <Emphasis Type="Italic">P. peruviana</Emphasis>, new natural hosts of <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis>

Tomato chlorosis virus causes yellow leaf disorder epidemics in many countries worldwide. Plants of Physalis ixocarpa showing abnormal interveinal yellowing and plants of Physalis peruviana showing mild yellowing collected in the vicinity of tomato crops in Portugal were found naturally infected with ToCV. Physalis ixocarpa and P. peruviana were tested for susceptibility to ToCV by inoculation with Bemisia tabaci, Q biotype. Results confirmed that ToCV is readily transmissible to both species. The infection was expressed in P. ixocarpa by conspicuous interveinal yellow areas on leaves that developed into red or brown necrotic flecks, while P. peruviana test plants remained asymptomatic. Infected plants of both P. ixocarpa and P. peruviana served as ToCV sources for tomato infection via B. tabaci transmission. This is the first report of P. ixocarpa and P. peruviana as natural hosts of ToCV.     

<Emphasis Type="Italic">Helicobasidium mompa</Emphasis> isolates from sweet potato in continuous monoculture fields

 Isolates of the violet root rot fungus Helicobasidium mompa were collected from herbaceous and tree plants. Their host preference was studied by inoculation experiments using carrots, sweet potatoes, and apple stocks. It was found that sweet potato isolates from Kyushu produced infection cushions on carrots and sweet potatoes but not on apple stocks. Other isolates did not show host preference. Sweet potato isolates were also characterized by ready hyphal mass (sclerotium) production. They were thought to have adapted to the habitat with high disturbance by annual tillage. Received: July 15, 2002 / Accepted: September 26, 2002     

Suppression of Fusarium wilt of spinach with hypovirulent binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis>

 Four isolates of hypovirulent binucleate Rhizoctonia (HBNR) were evaluated for their ability to control Fusarium wilt of spinach (FWS) caused by Fusarium oxysporum f. sp. spinaciae (FOS). Fourteen-day-old spinach seedlings grown in paper pots with HBNR-amended soil (1% w/w ground barley grain inoculum) were transferred to artificially pathogen-infested soil. Treatments with HBNR isolates significantly (P = 0.05) reduced disease and discoloration severity by 56%–100% and 52%–100%, respectively. The numbers of colony-forming units of FOS per gram fresh weight in petioles or roots were reduced significantly (P = 0.01) in the plants treated with HBNR. HBNR isolates were well reisolated from the roots inside paper pots where they were inoculated, whereas inconsistent colonization of HBNR was recorded from the roots outside paper pots where only pathogen was inoculated. Root extracts from HBNR-treated and pathogen-challenged plants significantly inhibited germination and germling length of FOS. The fresh weight of spinach leaves in the HBNR-treated plants increased significantly (P = 0.01), as much as 53%–63%, over the untreated and pathogen-challenged plants. This is the first report of biocontrol of FWS by HBNR. Received: July 18, 2002 / Accepted: October 22, 2002 Acknowledgments We are grateful to Dr. Komada for providing nonpathogenic Fusarium F13. The senior author (A.M.) thanks the Ministry of Education, Culture, Sports, Science, and Technology (Monbukagakusho) Japan, for financial assistance.     

Occurrence of <Emphasis Type="Italic">Phaeoacremonium</Emphasis> spp. and <Emphasis Type="Italic">Phaeomoniella chlamydospora</Emphasis> in grape propagation materials and young grapevines

During a 6-year study, grapevine propagation materials and young grapevines were analysed to evaluate the presence of internal wood discolouration and the occurrence of fungal species involved in Petri disease. The intensity of wood discolouration increased with the ageing of the plants. The maximum incidence of dark streaks was observed in the rootstock while necrosis originating from buds or nodes were notably present in the trunk and cordon of older vines. In contrast, the highest levels of brown-red halo symptoms, defined as discoloured areas around the pith, were recorded in the early growth stages. Phaeoacremonium spp. and Phaeomoniella chlamydospora were usually isolated from the rooted-grafts and the 3-year old plants, respectively. The number of infected grapevines increased with age. Most of the P. chlamydospora strains were isolated from dark streaks or dots, while Phaeoacremonium spp. were detected in brown-red halo symptoms and other symptomatic or asymptomatic wood. The greatest incidence of the two fungal taxa was recorded in the lower parts of the grapevine, including the roots and rootstock.