Development of specific primers for Fusarium oxysporum causing damping off of Lilium formolongi.

Primers specific for the hypothetical forma specialis of Fusarium oxysporum were designed to amplify DNA from this pathogenic fungus that infects plants including lilies. The F. oxysporum sequence between the transposal elements han and hop was used for primer design. Three primer pairs designed from this region were confirmed as specific for 24 isolates of F. oxysporum pathogenic to lilies, except for one pathogenic isolates as extraordinary. No amplification was observed from F. oxysporum non-pathogenic to lily, from 12 forma specialis, and 14 fungi and oomycetes concerned with Liliaceae plants. We propose that specific primers designed from this region will be useful to detect isolates of F. oxysporum that are pathogenic to lilies.

Characterization of a new carmovirus isolated from an Adonis plant.

An isometric virus was isolated from a cultivated Adonis plant (A. ramosa). The purified virus particle is 28 nm in diameter and is composed of a single coat protein and a single RNA genome of 3,991 nucleotides. Sequence analysis showed that the virus is closely related to carnation mottle virus. The virus was used to mechanically infect healthy A. ramosa plants, resulting in mosaic and leaf curl symptoms; however, attempts to inoculate carnation plants did not result in infection. We propose the virus as a new carmovirus and have named it adonis mosaic virus (AdMV).

Effect of Soil Inoculum Density and Temperature on the Incidence of Cucumber Black Root Rot.

The effects of the density of Phomopsis sclerotioides in soil and other environmental factors on black root rot of cucumber were investigated. Cucumber plants were grown in soil containing P. sclerotioides at 1, 10, 100, and 1000 CFU/g. Wilt incidence from 3 to 7 weeks after transplanting was strongly correlated with P. sclerotioides density in soil (P < 0.05). Root rot of squash rootstock occurred in soil with very low inoculum densities (0.1 CFU/g), and was strongly related to P. sclerotioides density (Y = -0.3x + 1.2, R2 = 0.743, P < 0.05) at 8 weeks after transplanting. Cucumber plants showed wilt symptoms in soil containing 1 CFU/g. Wilt symptoms in cucumber plants occurred 4 to 7 days earlier in soil at 22°C than in soil at 27 or 17°C. Root rot development could be predicted from the density of P. sclerotioides in soil and soil temperature. However, further studies on the effects of other environmental factors are required to test the linear model in commercial fields. This information is essential for determining the threshold pathogen density at which most control techniques, particularly those other than soil disinfection, will be effective.

Controlling Rhizoctonia Damping-off of Chinese Mustard by Using Endomycorrhizal Rhizoctonia spp. Isolated from Orchid Mycorrhizae.

Inoculation of hypovirulent Rhizoctonia spp. has been recognized as an effective strategy for protecting plants against damping-off caused by pathogenic Rhizoctonia spp. In this study, endomycorrhizal Rhizoctonia spp. isolated from fungal pelotons in orchid plants were used for controlling Rhizoctonia damping-off of Chinese mustard. According to phylogenetic analysis and anastomosis group (AG) determination, the virulence of three isolates of multinucleate Rhizoctonia solani in AG-6; eight isolates of binucleate Rhizoctonia in AG-A, AG-B, AG-G, AG-P, and AG-R; and two isolates of binucleate R. repens were evaluated using test plants. All isolates, except that in AG-R, caused low disease severity in 10-day-old radish (0.10 to 0.61), cucumber (0.28 to 0.54), and Chinese mustard (0.18 to 0.65). By contrast, pathogenic isolates in AG-4 killed almost all test plants with symptoms of collapsed hypocotyl and wilted leaves (0.88 to 0.96). Of the 13 endomycorrhizal Rhizoctonia isolates assessed, AG-P isolates Cno10-3 and CalS1-2 provided 91 and 100% protection, respectively, against R. solani AG-4 in 26-day-old Chinese mustard. This study revealed that endomycorrhizal Rhizoctonia spp. in orchid have the potential to biologically control damping-off of Chinese mustard.

Widespread Occurrence of Pythium arrhenomanes Pathogenic to Rice Seedlings Around Japanese Rice Fields.

In Japan, rice seedlings grown in nurseries and used for transplanting are subject to a damping-off disease caused by Pythium spp. In this study, 148 isolates of Pythium spp. were obtained from rice seedlings in 39 locations of northern Japan. Among the isolates, 137 were identified as Pythium arrhenomanes using polymerase chain reaction (PCR) with species-specific primers, DNA sequencing analyses of the internal transcribed regions of ribosomal DNA, and the morphologies of oogonia, antheridia, oospores, and zoosporangia. Inoculation tests showed that the isolates identified as P. arrhenomanes were pathogenic to rice seedlings and parasitic to southern crabgrass with only minor damage. P. arrhenomanes was reisolated from the roots of both rice seedlings and southern crabgrass. Poaceae weeds, hosts of Pythium spp., grow in and around nurseries and in ridges surrounding rice fields. To detect Pythium spp., 188 Poaceae weeds were collected from 37 locations in Akita Prefecture. P. arrhenomanes was frequently detected in 164 weed roots from all locations by PCR using species-specific primers. Thus, we determined that P. arrhenomanes exists in and around rice seedling nurseries and rice fields, and that it is much more widely distributed than previously recognized in northern Japan.

Draft Genome Sequence of the Plant-Pathogenic Soil Fungus Rhizoctonia solani Anastomosis Group 3 Strain Rhs1AP.

The soil fungus Rhizoctonia solani is a pathogen of agricultural crops. Here, we report on the 51,705,945 bp draft consensus genome sequence of R. solani strain Rhs1AP. A comprehensive understanding of the heterokaryotic genome complexity and organization of R. solani may provide insight into the plant disease ecology and adaptive behavior of the fungus.

Characterization of a Basidiomycete fungus from stored sugar beet roots.

Eighteen isolates from sugar beet roots associated with an unknown etiology were characterized based on observations of morphological characters, hyphal growth at 4-28 C, production of phenol oxidases and sequence analysis of internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA (rDNA). The isolates did not produce asexual or sexual spores, had binucleate hyphal cells with clamp connections, grew 4-22 C with estimated optimal growth at 14.5 C and formed a dark brown pigment on potato dextrose or malt extract agar amended with 0.5% tannic acid. Color changes observed when solutions of gum guiac, guiacol and syringaldzine were applied directly to mycelium grown on these media indicated that all isolates produced phenol oxidases. Sequences of ITS and LSU regions on the rDNA gene from 15 isolates were 99.2-100% identical, and analysis of sequence data with maximum likelihood and maximum parsimony suggest that the isolates from sugar beet roots are phylogenetically related to Athelia bombacina, Granulobasidium vellereum and Cyphella digitalis. High statistical support for both loci under different criteria confirmed that Athelia bombacina was consistently the closest known relative to the sugar beet isolates. Additional taxonomic investigations are needed before species can be clarified and designated for these isolates.

Seasonal Prevalence of Species of Binucleate Rhizoctonia Fungi in Growing Medium, Leaf Litter, and Stems of Container-Grown Azalea.

Rhizoctonia web blight is an annual problem on container-grown azalea (Rhododendron spp.) in the southern and eastern United States but little is documented about the distribution or persistence of Rhizoctonia spp. in container-grown azalea. Sixty web-blight-damaged azalea plants ('Gumpo White') were collected in August 2005 and 2006 and arranged in a completely randomized design on an outdoor irrigation pad. A nylon mesh bag containing 30 necrotic leaves collected from web-blight-damaged 'Gumpo White' azalea plants were placed on the surface of the medium under the plant canopy in each container to simulate leaf litter. Ten plants were destructively sampled into eight zones by dividing stems into three zones (lengths of 0 to 2, 4 to 6, and 9 to 15 cm above the medium surface), bagged leaves into one leaf litter zone, and the medium into four zones (three horizontal layers: 1 to 3, 3 to 7, and 7 to 10 cm below the medium surface, with the middle layer further divided by removing the central 7.5-cm-diameter core) in December, February, and May. Only the three stem zones were sampled from 10 plants in early and late June and late July. Of 8,940 total isolations, 3,655 fungi with morphological characteristics of a Rhizoctonia sp. were recovered. Percent recovery differed from the eight zones (P < 0.0001) but did not differ between years (P = 0.3950) and sampling times (P = 0.1896). Frequency of recovery of Rhizoctonia spp. was highest from the lower stem and the leaf litter, and decreased with distance from the leaf litter. Recovery from stems over the six sample times was analyzed separately. Percent recovery differed between stem zones (P < 0.0001), sample times (P = 0.0478), and experiment years (P < 0.0001). In both years, mean recovery of Rhizoctonia spp. was higher from the lower stem and decreased with distance to the upper stem layer. From a subsample of 145 isolates, 95.1% were identified as binucleate Rhizoctonia (BNR) anastomosis groups (AGs)-A, -G, -K, -R, -S, and -U (-P), and 2.8 and 2.1% were Rhizoctonia solani AG-2 and an uncultured Laetisaria sp., respectively. Based on frequency analysis, recovery of BNR AGs differed by plant zone (P < 0.0001) but not over sample times (P = 0.4831). The six AGs of BNR are the predominant Rhizoctonia fungi occupying the habitat niches in container-grown azalea, with little change in population frequency and composition from fall to summer; thus, BNR pathogenic and nonpathogenic to azalea have established a mixed Rhizoctonia community on container-grown azalea.

An In Planta Method for Assessing the Role of Basidiospores in Rhizoctonia Foliar Disease of Tomato.

A tomato (Solanum lycopersicum) foliar blight disease of unknown etiology was observed in North Carolina (NC) during 2005 to 2006. Symptoms included necrotic lesions and blighted leaves, with signs of white mycelial growth on abaxial leaf surfaces. The morphology of isolates from symptomatic leaves was consistent with that of Rhizoctonia solani. Because the pattern of symptom expression suggested that basidiospores were the primary inoculum source, Koch's postulates were fulfilled using a method to generate basidiospores in planta. Isolates were characterized by morphology, DNA sequence analysis, hyphal anastomosis, and somatic hyphal interactions. Phylogenetic analyses and hyphal anastomosis criteria support placement of the isolates in R. solani anastomosis group 3 (AG-3). Tomato foliar blight isolates from NC form a single phylogenetic group with tomato isolates of R. solani AG-3 from Japan and are more closely related to R. solani AG-3 isolates from potato than tobacco. Isolates exhibited both compatible and incompatible hyphal interactions when paired in vitro. To our knowledge, this is the first detailed report of tomato foliar blight caused by R. solani AG-3 in North America. A comprehensive description of the technique employed for producing basidiospores is presented with potential utility for understanding foliar disease etiology in other Rhizoctonia pathosystems.

Heterokaryon formation in Thanatephorus cucumeris (Rhizoctonia solani) AG-1 IC.

Approximately 50 single-basidiospore isolates (SBIs) obtained from each of 16 field isolates of Thanatephorus cucumeris AG-1 IC were examined for heterokaryon formation. All SBIs obtained from each field isolate were divided into two mating groups (SBIs-M1 and SBIs-M2), and tufts of mycelia were formed in the contact zone between colonies of paired SBIs-M1 and -M2 based on 0.5 % charcoal agar medium. Tufts were produced from all possible pairing between SBIs from non-parental field isolates. Hyphal anastomosis reactions indicated no cell death and random cell death at the contact cell, and was not related to tuft formation. AFLP phenotypes of SBIs from each field isolate were not identical to each other and were different from their parental field isolate. AFLP phenotypes of the tuft isolates formed from SBIs-M1 and SBIs-M2 from each field isolate were heterokaryotic. Moreover, several SBIs also formed tufts with their parental and non-parental field isolates. AFLP phenotypes of these tuft isolates suggested that they were all heterokaryotic. Results of these experiments suggest that T. cucumeris AG-1 IC is heterothallic and bipolar, and that genetic exchange can occur between homokaryotic and heterokaryotic isolates (Buller phenomenon).

Heterokaryon formation in Thanatephorus cucumeris anastomosis group 2-2 IV.

Thirty single basidiospore isolates (SBIs) obtained from four field isolates of the basidiomycete fungus Thanatephorus cucumeris AG 2-2 IV were examined for heterokaryon formation. SBIs of three of four field isolates (Rh509, 92155 and R94) did not produce a tuft of mycelium in the hyphal interaction zone between paired isolates on 2% charcoal agar. Field isolates Rh509, 92155 and R94 indicated no death of interacting mycelium with their progenies on glass slide and microscopic examination. AFLP (amplified fragment length polymorphism) phenotypes of parent and their SBIs were identical. Field isolates Rh509, 92155 and R94 and their SBIs were homothallic. SBIs obtained from field isolate SA-1 were grouped into two mating types (SBI-M1 and SBI-M2), and a tuft of mycelium was formed between paired SBIs-M1 and -M2. SBIs of field isolate SA-1 indicated that no death and death of interacting mycelium were randomly observed. AFLP phenotypes among SBIs of isolate SA-1 were not identical and were also different from their parent isolate. AFLP phenotypes of tuft mycelia produced between heterothallic SBI-M1 and -M2 were heterokaryotic. The mating system of field isolate SA-1 and its SBIs was heterothallic. Both SBIs-M1 and -M2 further produced tuft mycelium with homothallic field isolates and their SBIs. AFLP banding patterns suggested that tuft mycelium was heterokaryotic produced from between heterothallic and homothallic isolates. Results from these experiments clarified that both homothallic and heterothallic isolates exist in population of T. cucumeris AG 2-2 IV, and that genetic exchange can occur between homothallic and heterothallic isolates.

A New Subgroup of Rhizoctonia AG-D, AG-D III, Obtained from Japanese Zoysia Grass Exhibiting Symptoms of a New Disease.

Isolates of an unidentified Rhizoctonia sp. (UN isolates) were obtained from Japanese zoysia grass (Zoysia japonica Steud) that exhibited symptoms of a new sheath rot disease. UN isolates were binucleate and showed hyphal fusion with tester isolates of Rhizoctonia anastomosis group (AG)-D. Those isolates were compared with isolates of subgroups I and II of Rhizoctonia AG-D based on cultural morphology, hyphal growth rate at different temperatures, anastomosis frequency, pathogenicity, and sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA genes (rDNA-ITS region). The mycelial color of UN isolates was light yellow which differs from AG-D I but is similar to AG-D II. Sclerotia of UN isolates were dark brown in color and larger in size (1 to 3 mm in diameter) than those of AG-D subgroup I (1 mm in diameter), whereas isolates of AG-D II produced white mycelial clamps 4 to 5 mm in size. Hyphal growth rate of UN isolates was slower than that of two AG-D subgroups at several temperatures, especially 25°C. In pathogenicity tests on Japanese zoysia grass, UN isolates showed moderate disease severity and lower pathogenicity than isolates of AG-D subgroups I and II. Sequences of the rDNA-ITS region within UN isolates were almost homologous, but had lower homology with subgroups AG-D I or II. Phylogenetic trees constructed using ITS sequences showed that UN isolates formed an individual cluster that differed from the clusters of the two subgroups. We propose that UN isolates are a new subgroup of Rhizoctonia AG-D, subgroup III, and the name of the disease is "spring-rot" on Japanese zoysia grass.

Brown Ring Patch: A New Disease on Bentgrass Caused by Waitea circinata var. circinata.

Isolates of an unidentified Rhizoctonia sp. (NP isolates), obtained from creeping bentgrass (Agrostis stolonifera var. palustris) in Japan that exhibited symptoms of a new disease, were compared with isolates of three varieties of Waitea circinata var. oryzae, var. zeae, and var. circinata. NP isolates also were compared with isolates of R. oryzae obtained from creeping bent-grass exhibiting white patch-like symptoms (RW isolates). The color and size of sclerotia, color of mycelia, and pigment deposition of NP isolates was similar to that of RW isolates and W. circinata var. circinata, but distinctly different from W. circinata var. oryzae and W. circinata var. zeae. The optimal temperature for hyphal growth of NP isolates, RW isolates, and W. circinata var. circinata was 28°C, and for W. circinata var. oryzae and W. circinata var. zeae was 30°C. Pathogenicity tests on creeping bentgrass showed that the severity of disease caused by NP isolates, RW isolates, and W. circinata var. circinata was greater than with W. circinata var. oryzae, but lower than with W. circinata var. zeae. No significant differences in symptom expression were apparent among NP isolates, RW isolates, and W. circinata var. circinata. A phylogenic tree, obtained using the results of random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), showed that isolates of W. circinata var. oryzae and W. circinata var. zeae separated into individual clusters, while NP isolates, RW isolates, and W. circinata var. circinata clustered together. The lengths of the rDNA internal transcribed spacer (ITS) region of NP isolates, RW isolates, and W. circinata var. circinata were identical but smaller than those of W. circinata var. oryzae and W. circinata var. zeae. Restriction fragment length polymorphism (RFLP) analysis of the rDNA-ITS region, using three enzymes (HapII, HinfI, and MboI), also showed that NP isolates were the same as RW isolates and W. circinata var. circinata, but different from W. circinata var. oryzae and W. circinata var. zeae. Based on these results, the NP isolates causing a new disease on bentgrass are W. circinata var. circinata, and that RW isolates are also W. circinata var. circinata but not R. oryzae. We propose that the name of the disease on creeping bentgrass caused by W. circinata var. circinata is brown ring patch.

Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass.

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3-10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.

Protective effects of flavonoids on the cytotoxicity of linoleic acid hydroperoxide toward rat pheochromocytoma PC12 cells.

The protective effects of nine flavonoids, including apigenin, eriodictyol, 3-hydroxyflavone, kaempherol, luteolin, quercetin, rutin, and taxifolin (Table 1), on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat pheochromocytoma PC12 cells were examined. The cytotoxicity was assessed by the trypan blue exclusion test and so-called MTT assay. When cells were preincubated with each flavonoid prior to LOOH exposure, quercetin, 3-hydroxyflavone, or luteolin decreased LOOH cytotoxicity toward undifferentiated cells, while only luteolin decreased efficiently LOOH cytotoxicity toward differentiated cells. On the other hand, when cells were coincubated with each flavonoid and LOOH, kaempherol, eriodictyol, quercetin, 3-hydroxyflavone, luteolin, or taxifolin decreased LOOH cytotoxicity toward undifferentiated and differentiated cells. On both preincubation prior to LOOH exposure and coincubation with LOOH, luteolin acted as the most efficiently protective agent against LOOH cytotoxicity. Further, these flavonoids showed protective effects on coincubation rather than preincubation. Flow cytometry using the fluorescence probe 2',7'-dichlorofluorescin diacetate revealed that LOOH increases the intracellular level of reactive oxygen species in undifferentiated cells in a dose-dependent manner, and that desferrioxamine mesylate suppresses the LOOH-induced increase in the level. These flavonoids suppress the LOOH-induced increase. Further, the protective effect of flavonoids on LOOH cytotoxicity correlates with the suppression of the LOOH-induced increase. These results suggest that such flavonoids are beneficial for neuronal cells under oxidative stress.

Flavonoids suppress the cytotoxicity of linoleic acid hydroperoxide toward PC12 cells.

The suppressive effect of flavonoids on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat phenochromocytoma PC12 cells was examined. The extent of cytotoxicity was shown on the basis of % survival determined by the trypan blue exclusion test. On preincubation of cells with either 3-hydroxyflavone, quercetin, or luteolin prior to LOOH exposure, the cytotoxicity was considerably suppressed. In contrast, on coincubation of cells with either eriodictyol, quercetin, kaempherol, luteolin, or 3-hydroxyflavone and LOOH, it was markedly suppressed. Regardless of incubation conditions, quercetin, 3-hydroxyflavone, and luteolin were thus more effective as protective agents against the cytotoxicity than the other flavonoids. These flavonoids further showed a suppressive effect on coincubation rather than on preincubation. These results suggest that such flavonoids are beneficial for cells under oxidative stress.