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     

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.     

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.     

Leaf spot of <Emphasis Type="Italic">Aster savatieri</Emphasis> (Makino) Kitam. caused by <Emphasis Type="Italic">Septoria chrysanthemella</Emphasis> Saccardo

 In May 1998 leaf spot caused by Septoria chrysanthemella was found on Aster savatieri in Kanagawa Prefecture, Japan. This is the first report of leaf spot on A. savatieri caused by S. chrysanthemella. Received: September 13, 2002 / Accepted: October 18, 2002 Acknowledgments The authors thank Dr. T. Kobayashi, formerly of Tokyo University of Agriculture, for his advice on identifying the fungus.     

Induced systemic resistance of Chinese cabbage to bacterial leaf spot and <Emphasis Type="Italic">Alternaria </Emphasis>leaf spot by the root endophytic fungus, <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis>

 The root endophytic fungus Heteroconium chaetospira isolate OGR-3 was tested for its ability to induce systemic resistance in Chinese cabbage against bacterial leaf spot caused by Pseudomonas syringae pv. maculicola and Alternaria leaf spot caused by Alternaria brassicae of the foliar diseases. Chinese cabbage seedlings planted in soil infested with an isolate of H. chaetospira were incubated in a growth chamber for 32 days. The first to fourth true leaves of the seedlings were challenge-inoculated with P. syringae pv. maculicola or A. brassicae. Chinese cabbage planted in soil infested with H. chaetospira showed significant decreases in the number of lesions of bacterial leaf spot or Alternaria leaf spot when compared to the control plants not treated with H. chaetospira. The results indicated that colonization of roots by H. chaetospira could induce systemic resistance in Chinese cabbage and reduce the incidence of bacterial leaf spot and Alternaria leaf spot. Received: April 24, 2002 / Accepted: August 9, 2002     

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.     

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.     

Induced suppressiveness to<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp.<Emphasis Type="Italic">radicis lycopersici</Emphasis> in rockwool substrate used in closed soilless systems

Tomatoes grown in soilless systems can be seriously damaged byFusarium oxysporum Schlect f.sp.radicis lycopersici (Forl) causing Fusarium crown and root rot (FCRR). FCRR suppression can be achieved through the use of chemicals, selected substrates, composts and artificially introduced antagonistic microorganisms. This study evaluated the natural capacity of a used rockwool to suppress FCRR infections. New and used rockwool, sampled from closed soilless systems, was either autoclaved or not, either artificially inoculated withForl or not and, finally, sown with tomato seeds cv. ‘Cuore di Bue’. The effects of autoclaved/non-autoclaved and used/new rockwool on FCRR incidence were assessed by evaluating the symptoms of crown rot on the root — shoot transition zone of tomato seedlings. Non-autoclaved and inoculated used rockwool significantly reduced FCRR incidence when compared with non-autoclaved and inoculated new rockwool. Autoclaved and inoculated used rockwool did not suppress FCRR, similarly to new and inoculated rockwool. These findings are in accordance with other research that, on a cucumber/Pythium host/pathogen complex in a closed rockwool soilless system, demonstrated the key role of resident microflora in suppressing the root rot disease. http://www.phytoparasitica.org posting Dec. 8, 2006.     

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.     

Cloning and characterization of pea apyrases: involvement of <Emphasis Type="Italic">PsAPY1 </Emphasis>in response to signal molecules from the pea pathogen <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis>

 Two nucleoside triphosphatase (NTPase) cDNA clones were isolated from a cDNA library of Pisum sativum L., cv. Midoriusui. The genes encoding the cDNAs were designated PsAPY1 and PsAPY2. PsAPY1 included the N-terminal amino acid sequence of an NTPase bound to pea cell wall. The phylogenic analysis indicated that PsAPY1 belongs to an NTPase subfamily responsive to environmental stimuli and that PsAPY2 belongs to a discrete subfamily, the physiological role of which is almost unknown. The adenosine triphosphatase activity of recombinant PsAPY1 was regulated by an elicitor and a suppressor from the pea pathogen Mycosphaerella pinodes. Based on these findings, we discuss the role of NTPases in response to biological stresses. Received: May 27, 2002 / Accepted: July 31, 2002     

Differential interactions of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> with <Emphasis Type="Italic">Brassica napus</Emphasis> detected with molecular and histological techniques

The differential interactions of V. longisporum (VL) and V. dahliae (VD) on the root surface and in the root and shoot vascular system of Brassica napus were studied by confocal laser scanning microscopy (CLSM), using GFP tagging and conventional fluorescence dyes, acid fuchsin and acridin orange. VL and VD transformants expressing sGFP were generated by Agrobacterium-mediated transformation. GFP signals were less homogenous and GFP tagging performed less satisfactory than the conventional fluorescence staining when both were studied with CLSM. Interactions of both pathogens were largely restricted to the root hair zone. At 24 h post-inoculation (hpi), hyphae of VL and VD were found intensely interwoven with the root hairs. Hyphae of VL followed the root hairs towards the root surface. At 36 hpi, VL hyphae started to cover the roots with a hyphal net strictly following the grooves of the junctions of the epidermal cells. VL started to penetrate the root epidermal cells without any conspicuous infection structures. Subsequently, hyphae grew intracellularly and intercellularly through the root cortex towards the central cylinder, without inducing any visible plant responses. Colonisation of the xylem vessels in the shoot with VL was restricted to individual vessels entirely filled with mycelium and conidia, while adjacent vessels remained completely unaffected. This may explain why no wilt symptoms occur in B. napus infected with VL. Elevated amounts of fungal DNA were detectable in the hypocotyls 14 days post-inoculation (dpi) and in the leaves 35 dpi. Root penetration was also observed for VD, however, with no directed root surface growth and mainly an intercellular invasion of the root tissue. In contrast to VL, VD started ample formation of conidia on the roots, and was unable to spread systemically into the shoots. VD did not form microsclerotia in the root tissue as widely observed for VL. This study confirms that VD is non-pathogenic on B. napus and demonstrates that non-host resistance against this fungus materializes in restriction of systemic spread rather than inhibition of penetration.     

Molecular Characterization of <Emphasis Type="Italic">Bean yellow mosaic virus </Emphasis>from Vegetatively Propagated Dwarf <Emphasis Type="Italic">Gentiana </Emphasis>Plants

The causative virus (isolate No. 4) of gentian (Gentiana spp.) mosaic, which had been identified previously as Clover yellow vein virus (C1YVV) on the basis of host range and serological reactions, was re-identified as Bean yellow mosaic virus (BYMV) on the basis of the nucleotide sequences of the gene for the coat protein (CP) and the 3′-noncoding region, as well as the predicted amino acid sequence of CP. Received 16 April 2002/ Accepted in revised form 19 June 2002     

<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     

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     

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.     

Long-term storage and survival structure of three species of <Emphasis Type="Italic">Phytophthora </Emphasis>in water

 Cultures of Phytophthora cinnamomi, P. parasitica, and P. palmivora remained viable in water at room temperature for periods ranging from 6 to 23 years. The colonies that developed from the stored cultures were thin-walled and spherical, ranging from 19.2 to 30.0 μm in diameter. The survival structures are thought to be small chlamydospores produced in the absence of adequate nutrition and aeration. Received: October 7, 2002 / Accepted: January 8, 2003 Acknowledgment I thank Dr. Michael L. Parsons for assistance in preparing the photograph.     

Addition of <Emphasis Type="Italic">Pestalotiopsis </Emphasis>spp. to leaf spot pathogens of Japanese persimmon

 In June 1996, a leaf spot disease widely occurred in Japanese persimmon (Diospyros kaki) orchards in Tottori Prefecture, Japan. The main diagnostic symptom was ring spot on the leaves and calyxes of young fruits; in severe cases, lesions developed on more than half of the area of the leaf, resulting in early defoliation. Based on morphological and pathological studies of the isolated fungi, it was shown that Pestalotiopsis longiseta, P. glandicola, P. acaciae, and P. crassiuscula were responsible for the diseases. These fungi, except P. longiseta, were found to be new pathogens of the disease. Received: May 20, 2002 / Accepted: July 25, 2002