Invasive Haemophilus influenzae disease among adults in Japan during 2014-2018.

PURPOSE: We describe the epidemiology of invasive Haemophilus influenzae disease (IHD) among adults in Japan. METHODS: Data for 200 adult IHD patients in 2014-2018 were analyzed. The capsular type of H. influenzae was determined by bacterial agglutination and polymerase chain reaction (PCR), and non-typeable Haemophilus influenzae (NTHi) was identified by PCR. RESULTS: The annual incidence of IHD (cases per 100,000 population) was 0.12 for age 15-64 years and 0.88 for age ≥ 65 years in 2018. The median age was 77 years, and 73.5% were aged ≥ 65 years. About one-fourth of patients were associated with immunocompromising condition. The major presentations were pneumonia, followed by bacteremia, meningitis and other than pneumonia or meningitis (other diseases). The case fatality rate (CFR) was 21.2% for all cases, and was significantly higher in the ≥ 65-year group (26.1%) than in the 15-64-year group (7.5%) (p = 0.013). The percentage of cases with pneumonia was significantly higher in the ≥ 65-year group than in the 15-64-year group (p < 0.001). The percentage of cases with bacteremia was significantly higher in the 15-64-year group than in the ≥ 65-year group (p = 0.027). Of 200 isolates, 190 (95.0%) were NTHi strains, and the other strains were encapsulated strains. 71 (35.5%) were resistant to ampicillin, but all were susceptible to ceftriaxone. CONCLUSION: The clinical presentations of adult IHD patients varied widely; about three-fourths of patients were age ≥ 65 years and their CFR was high. Our findings support preventing strategies for IHD among older adults, including the development of NTHi vaccine.

[Lumbo-Peritoneal Shunt for Patients with Idiopathic Normal Pressure Hydrocephalus:Surgical Technique].

The SINPHONI-2 study(a group of Japanese prospective multicenter cohort studies on the treatment of idiopathic normal pressure hydrocephalus[iNPH])showed the safety and efficacy of lumboperitoneal(L-P)shunt surgery for iNPH. A total of 660 probable iNPH patients underwent L-P shunt surgery at our NPH center between April 2009 and March 2020(age: 77.3 ± 6.2 years). Our surgical technique includes 1)general anesthesia, 2)use of the original drape, 3)upward insertion of the spinal tube through L2/3 via a paramedian puncture in patients with highly deformed lumbar spines, 4)posterior placement of a Codman Hakim programmable valve with SiphonguardTM, 5)inclination of the table at a 35° angle without position change and re-sterilization, 6)laparotomy via rectal muscle splitting, and 7)oblique maneuvering the peritoneal tube from the upper lateral to the lower medial sector. During the first year after surgery, postoperative complications occurred in 14 of 172 patients (8.1%), including four patients with chronic subdural hematoma requiring evacuation (2.3%), three with spinal tube occlusion (1.7%), three with migration of the spinal tube, two with lower-limb numbness (1.2%), and two with abdominal tube occlusion. Our L-P shunt procedure seems to be generally acceptable considering the low number of complications.

Invasive Haemophilus influenzae type a infection and polyarthritis in a 72-year-old Japanese man: A case report.

Haemophilus influenzae is a small, nonmotile, non-spore-forming bacterium classified into 6 serotypes (a to f) and non-typeable strains that lack a capsule. Although H. influenzae serotype a (Hia) is prevalent in Canada, the United States, Brazil, Australia, across the African continent, and several other locations, it has not been reported in Japan thus far. Our case was of a 72-year-old Japanese man who sought medical consultation after presenting with chills, fever, and polyarthritis. Cultures of blood and synovial fluid from the left knee revealed H. influenzae infection. Diagnostic imaging showed poor contrast regions in both kidneys, fluid retention around both knee joints, the left shoulder joint, and both elbow joints. Subsequently, the patient was diagnosed with invasive H. influenzae infection accompanied by polyarthritis and renal infarction. 16S ribosomal RNA gene sequencing revealed that the bacterial strain was Hia. The patient was treated with antimicrobial agents and arthroscopic curettage. We present a case of invasive Hia infection accompanied by polyarthritis and renal infarction. To the best of our knowledge, this is the first case of Hia infection in Japan. The case is very rare considering that the disease occurred in an elderly patient who developed polyarthritis.

Complete Genome Sequence of a Japanese Clinical Isolate of Haemophilus influenzae Type a Strain TAMBA230.

Haemophilus influenzae causes severe infections such as pneumonia and meningitis. Here, we report the complete genome of H. influenzae type a strain TAMBA230, which was isolated in 2019 from a patient exhibiting bacteremia. This represents the first case in Japan of an H. influenzae type a strain associated with invasive infection.

T-Cell/Histiocyte-Rich Large B-Cell Lymphoma Presented as T-Lymphoid Hyperplasia Involving the Central Nervous System.

We herein report a case of T-cell/histiocyte-rich large B-cell lymphoma which initially presented as a self-limiting T-lymphoproliferative disorder involving multiple extranodal and extrapulmonary organs, such as the salivary gland, the liver, and the central nervous system. Repeated biopsies only revealed polyclonal T-lymphocytosis without the presence of atypical B-cells. Angiocentric cellular infiltration was absent, thus ruling out lymphomatoid granulomatosis. A recurrence in the lymphatic system finally revealed a small population of pathognomonic atypical B-cells, which led to the diagnosis. The clinical dilemma in the diagnosis and management of this indeterminate condition points to limitations in the current nosology.

Long-term outcome and neuroradiologic changes after multiple hippocampal transection combined with multiple subpial transection or lesionectomy for temporal lobe epilepsy.

OBJECTIVE: Multiple hippocampal transection (MHT) is a surgical procedure developed to avoid postoperative memory decline. Its efficacy has been documented in only a few small series with relatively short observation periods. We prospectively evaluated the long-term seizure and cognitive outcomes of MHT combined with multiple subpial transection or lesionectomy (MHT + MST/L). Moreover, we quantitatively evaluated the structural and metabolic neuroradiologic changes after the procedure to elucidate the anatomofunctional correlates of memory preservation. METHODS: Twenty-four patients underwent MHT + MST/L for treatment of drug-resistant mesial temporal lobe epilepsy (mTLE) and were followed for more than 5 years. Indications for the procedure were the following: (1) verbally dominant-sided surgery in patients with a radiologically normal hippocampus or normal/near normal memory, and (2) surgery for patients with concomitant epileptic activity on the contralateral side, that is, when the surgery was considered a high risk for severe postoperative memory decline. Seizure outcome was evaluated using Engel's classification 1, 2, and 3 years after surgery, and at the last visit (LV). Three subgroups were evaluated as well: magnetic resonance imaging (MRI) negative (MN), hippocampal sclerosis (HS), and normal hippocampus with extrahippocampal lesion (NHEL). The long-term cognitive outcome was followed through to LV in patients who underwent verbally dominant-sided surgery. Hippocampal volume (HV), diffusion tensor parameters (DTP), and glucose utilization (GU) were determined from MRI and fluorodeoxyglucose-positron emission tomography (FDG-PET) studies performed before and >6 months after surgery. RESULTS: Whereas the rate of Engel class I as a whole was 71% at 1 year and 67% at LV, the rates in the MN, HS, and NHEL groups were 60%, 67%, and 100% at 1 year, respectively, and 70%, 56%, and 80% at LV, respectively. Memory indices after verbally dominant-sided surgery transiently declined at 1 month but recovered to and remained at the preoperative level through LV. The HV, DTP of the fornix, and GU of the temporal lobe on the treated side showed pathologic changes even when the transiently declined memory indices had recovered to the preoperative level. SIGNIFICANCE: The long-term outcome for complex partial seizures after MHT + MST/L was comparable to that seen after anterior temporal lobectomy. The long-term cognitive outcome was favorable, even for patients with a high risk of severe postoperative memory decline. MHT + MST/L may be a treatment option for mTLE in which resective surgery carries a risk of postoperative memory decline, particularly in patients without MRI lesion. A discrepancy between the preserved memory and the pathologic neuroradiologic changes indicates the necessity for further studies including functional MRI.

Selective Patch Angioplasty and Intraoperative Shunting in Carotid Endarterectomy: A Single-Center Review of 141 Procedures.

OBJECTIVE: Open surgical treatment of carotid artery stenosis, namely, carotid endarterectomy (CEA), has evolved since its inception in 1953. Despite improvements in the treatment of carotid occlusive disease through technological and surgical innovations, the use of patch grafting in CEA's remains controversial. We evaluate the durability of the primary closure and the safety of selective shunting during carotid endarterectomy (CEA) as determined by intraoperative EEG and postoperative outcomes. METHODS: A consecutive series of CEA's performed by the senior author at a single academic medical center from 2001 to 2012 were reviewed. All cases were performed under continuous intraoperative electroencephalography (EEG). Patch angioplasty was used in cases where there was tortuosity of the vessel within the region of the endarterectomy and narrow vessel diameter at the distal end of the arteriotomy. Shunting was used when intraoperative EEG showed a > 50% reduction in a waveform in any lead. Patients were evaluated for restenosis via imaging or ultrasound at six months and subsequently annual follow-up. RESULTS: One hundred and forty-one CEA's were performed on 132 (76 male, 56 female) patients with an average age of 71 years (range: 40-95 years). Four (3%) cases required patch angioplasty and three (2%) required intraoperative shunts. The cross-clamp time ranged from 22 to 74 minutes, and the duration increased with the use of shunts and patches. Complications were rare and included recurrent stenosis (n=2), postoperative transient ischemic attack (n=1), ischemic stroke in (n=1), temporary hypoglossal nerve weakness (n=2), temporary marginal mandibular nerve weakness (n=6), and neck hematoma (n=1). CONCLUSION: Intraoperative EEG data suggests that primary closure and selective shunting in CEA can result in outcomes comparable with routine patch angioplasty and shunting.

Involvement of the salicylic acid signaling pathway in the systemic resistance induced in Arabidopsis by plant growth-promoting fungus Fusarium equiseti GF19-1.

Plant growth-promoting fungi (PGPF) are effective biocontrol agents for a number of soil-borne diseases and are known for their ability to trigger induced systemic resistance (ISR). In this study, we investigated the mechanisms triggered by PGPF Fusarium equiseti GF19-1, which is known to increase pathogen resistance in plants, by using GF19-1 spores and the culture filtrate (CF) to treat the roots of Arabidopsis thaliana. Subsequently, the leaves were challenged with Pseudomonas syringae pv tomato DC3000 (Pst) bacteria. Arabidopsis plants treated with GF19-1 spores or the CF elicited ISR against the Pst pathogen, resulting in a restriction of disease severity and suppression of pathogen proliferation. Examination of ISR in various signaling mutants and transgenic plants showed that GF19-1-induced protection was observed in the jasmonate response mutant jar1 and the ethylene response mutant etr1, whereas it was blocked in Arabidopsis plants expressing the NahG transgene or demonstrating a disruption of the NPR1 gene (npr1). Analysis of systemic gene expression revealed that GF19-1 modulates the expression of salicylic acid (SA)-responsive PR-1, PR-2, and PR-5 genes. Moreover, transient accumulation of SA was observed in GF19-1-treated plant, whereas the level was further enhanced after Pst infection of GF19-1-pretreated plants, indicating that accumulation of SA was potentiated when Arabidopsis plants were primed for disease resistance by GF19-1. In conclusion, these findings imply that the induced protective effect conferred by F. equiseti GF19-1 against the leaf pathogen Pst requires responsiveness to an SA-dependent pathway.

Mouse in utero electroporation: controlled spatiotemporal gene transfection.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Enhanced defense responses in Arabidopsis induced by the cell wall protein fractions from Pythium oligandrum require SGT1, RAR1, NPR1 and JAR1.

The cell wall protein fraction (CWP) is purified from the non-pathogenic biocontrol agent Pythium oligandrum and is composed of two glycoproteins (POD-1 and POD-2), which are structurally similar to class III elicitins. In tomato plants treated with CWP, jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways are activated, and resistance to Ralstonia solanaceraum is enhanced. To dissect CWP-induced defense mechanisms, we investigated defense gene expression and resistance to bacterial pathogens in Arabidopsis thaliana ecotype Col-0 treated with CWP. When the leaves of Col-0 were infiltrated with CWP, neither visible necrosis nor salicylic acid (SA)-responsive gene (PR-1 and PR-5) expression was induced. In contrast, JA-responsive gene (PDF1.2 and JR2) expression was up-regulated and the resistance to R. solanaceraum and Pseudomonas syringae pv. tomato DC3000 was enhanced in response to CWP. Such CWP-induced defense responses were completely compromised in CWP-treated coi1-1 and jar1-1 mutants with an impaired JA signaling pathway. The induction of defense-related gene expression after CWP treatment was partially compromised in ET-insensitive ein2-1 mutants, but not in SA signaling mutants or nahG transgenic plants. Global gene expression analysis using cDNA array also suggested that several other JA- and ET-responsive genes, but not SA-responsive genes, were up-regulated in response to CWP. Further analysis of CWP-induced defense responses using another eight mutants with impaired defense signaling pathways indicated that, interestingly, the induction of JA-responsive gene expression and enhanced resistance to two bacterial pathogens in response to CWP were completely compromised in rar1-1, rar1-21, sgt1a-1, sgt1b (edm1) and npr1-1 mutants. Thus, the CWP-induced defense system appears to be regulated by JA-mediated and SGT1-, RAR1- and NPR1-dependent signaling pathways.

High level expression of a virus resistance gene, RCY1, confers extreme resistance to Cucumber mosaic virus in Arabidopsis thaliana.

A coiled coil-nucleotide binding site-leucine rich repeat-type resistance gene, RCY1, confers resistance to a yellow strain of Cucumber mosaic virus, CMV(Y), in Arabidopsis thaliana ecotype C24. Resistance to CMV(Y) in C24 is accompanied by a hypersensitive response (HR) that is characterized by the development of necrotic local lesions at the primary infection sites. To further study the HR and resistance to CMV(Y) in ecotype Col-0, which is susceptible to CMV(Y), Col-0 were transformed with RCY1. Systemic spread of CMV(Y) was completely suppressed in RCY1-transformed Col-0 (Col::pRCY1 lines 2 to 6), whereas virulent strain CMV(B2) spread and multiplied systemically in these transgenic lines similar to that in wild-type Col-0. Interestingly, the resistant phenotype of Col::pRCY1 varied among the lines. In lines 3 and 6, in which levels of RCY1 transcript were similar to that in wild-type C24, the HR and resistance to CMV(Y) was induced. Line 4, which expresses moderately elevated levels of RCY1 transcript, exhibited moderately enhanced resistance compared with that in C24 or line 3. In contrast, lines 2 and 5, which highly overexpress the RCY1 gene, did not exhibit either visible lesions or a micro-HR on the inoculated leaves. Moreover, virus coat protein was not detected in either inoculated or noninoculated upper leaves of these two lines, suggesting that extreme resistance (ER) to CMV(Y) was induced by high levels of expression of RCY1. Furthermore, in transgenic lines expressing hemagglutinin (HA) epitope-tagged RCY1 (Col::pRCY1-HA), high levels of accumulation of RCY1-HA protein were also correlated with the ER phenotype. Global gene expression analysis in line 2, which highly overexpresses RCY1, indicated that expression of several defense-related genes were constitutively elevated compared with wild-type Col-0. Despite this, line 2 did not have enhanced resistance to other avirulent and virulent pathogens. Take together, constitutive accumulation of high levels of RCY1 protein appears to regulate the strength of RCY1-conferred resistance in a gene-for-gene manner and implies that ER and HR-associated resistance differ only in the strength of resistance.

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).

Syringomyelia caused by intrathecal remnants of oil-based contrast medium.

Oily contrast medium had been in use since the early 19th century as a radiographic agent for detecting spinal lesions and spinal cord tumors until the late 20th century. At that point computed tomography scanning and magnetic resonance imaging, or other hydrophilic contrast medium substituted for it. Adverse effects of oil-based dye, both acute and chronic, had been reported since the middle of the 20th century. In this paper the authors report the case of syringomyelia that seemed to be caused mainly by remaining oily contrast medium for 44 years. Syringomyelia secondary to adhesive arachnoiditis caused by oily contrast medium after a long period of time is well known. In the present case, however, surgery revealed only mild arachnoiditis at the level of syringomyelia as well as both solid and liquid remnants of contrast medium. Generally, cerebrospinal fluid (CSF) blockage due to an arachnoid adhesion is considered to cause syringomyelia following adhesive arachnoiditis. The authors speculated that in the present case syringomyelia was induced by a mechanism different from that in the previously reported cases; the oily contrast medium itself seems to have induced the functional block of CSF and impaired the buffer system of the intrathecal pressure. No reports on thoracic adhesive arachnoiditis and syringomyelia caused by oil-based dye referred to this mechanism in reviewing the literature.

The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals.

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.

The feedback phase of type I interferon induction in dendritic cells requires interferon regulatory factor 8.

Dendritic cells (DCs) produce type I interferons (IFNs) in greater amounts than other cells, but the mechanisms remain elusive. Here we studied the role of a transcription factor, IRF8, in DC induction of type I IFNs. Upon newcastle disease virus (NDV) infection, bone marrow-derived plasmacytoid and conventional DCs induced IFN transcripts, exhibiting two-phase kinetics. The second, amplifying phase represented an IFN feedback response that accounted for much of IFN protein production. Induction of second phase transcription required IRF8. Mouse cytomegalovirus (MCMV) and Toll-like receptor-mediated IFN induction in DCs also required IRF8. Chromatin immunoprecipitation analysis showed that IRF7, IRF8, and RNA polymerase II were recruited to the IFN promoters upon stimulation. Moreover, sustained RNA polymerase II recruitment to the promoters critically depended on IRF8. Together, these data indicate that IRF8 magnifies the second phase of IFN transcription in DCs by prolonging binding of basic transcription machinery to the IFN promoters, thereby playing a role in innate immunity.

Clinical characteristics and surgical management for juxtafacet cysts of the lumbar spine.

Retrospective analysis of 10 cases of resection of symptomatic lumbar juxtafacet cysts in nine patients (mean age 65.4 years) investigated the relationship between surgical method and progression of spinal spondylolisthesis or cyst recurrence. Patient characteristics, surgical methods, and postoperative course were reviewed. The most common preoperative symptom, painful radiculopathy, occurred in all cases, followed by motor weakness in five, sensory loss in four, and intermittent claudication in four. All patients underwent bilateral total (n = 6) or partial laminectomy (n = 4), with minimal (n = 3) or no (n = 7) facetectomy. Cysts were gross totally resected in eight cases and partially resected in two. Concomitant fixation was not performed. Painful radiculopathy, motor weakness, and sensory disturbance all resolved, resulting in good or excellent outcome in all patients. Postoperative symptomatic spondylolisthesis had not been noted at mean 52.1 months postoperatively. However, new juxtafacet cysts were later detected on the contralateral side to the initial lesion in two patients. Surgical removal of juxtafacet cysts is recommended for immediate symptomatic relief. Concomitant spinal fixation to prevent progression of spinal spondylolisthesis or cyst recurrence depends on cyst size, involvement of surrounding structures, degree of preoperative spondylolisthesis, and facet joint destruction.

Co-occurrence of Arum- and Paris-type morphologies of arbuscular mycorrhizae in cucumber and tomato.

Colonization by arbuscular mycorrhizal (AM) fungi was investigated in cucumber (Cucumis sativus), tomato (Lycopersicon esculentum) and Clethra barbinervis (Ericales) grown in field-collected soil known from previous studies to generate Paris-type arbuscular mycorrhizae in C. barbinervis. Spores of Paraglomus, Acaulospora, Glomus, and Gigaspora were found in the soil. Formation of hyphal coils and arbusculate coils of Paris-type mycorrhizae and of arbuscules of Arum-type mycorrhizae in roots raised in this soil in the growth chamber were compared with the detection of DNA of AM fungi from the same root systems using Glomales-specific primers. Only Paris-type mycorrhizae with extensive arbusculate coils developed in C. barbinervis, but cucumber and tomato developed both Paris- and Arum-types in the same root systems. Glomaceae and Archaeosporaceae and/or Paraglomaceae were detected strongly in the DNA from both cucumber and tomato roots, in which Arum-type mycorrhizae were observed. In contrast, DNA of Glomaceae was detected more sparingly in C. barbinervis, in which Paris-type mycorrhizae dominated. Acaulosporaceae and Gigasporaceae were strongly detected in the DNA from both C. barbinervis and tomato, whereas they were more weakly detected in cucumber. These results indicate that the morphology of colonization is strongly influenced by the selection of fungi to colonize the host plant from among those in the soil environment.

New Anastomosis Groups, AG-T and AG-U, of Binucleate Rhizoctonia spp. Causing Root and Stem Rot of Cut-Flower and Miniature Roses.

ABSTRACT Root and stem rot of cut-flower roses (Rosa spp.) was observed in commercial glasshouse-grown roses in 10 prefectures of Japan from 1998 through 2001. Binucleate-like Rhizoctonia spp. were isolated mainly from the disease plants. In all, 670 isolates were divided into two types based on cultural appearance; 168 isolates of light brown to brown type and 502 isolates of whitish type. A hyphal anastomosis reaction using representative isolates from each type revealed that the light brown to brown type belonged to anastomosis group G (AG-G), whereas the whitish type (AG-CUT) failed to anastomose with tester strains of binucleate Rhizoctonia AG-A through AG-S. Neither isolates of AG-G nor AG-CUT anastomosed with tester strains of a previously reported unknown AG (AG-MIN) of binucleate Rhizoctonia spp. collected from miniature roses. In pathogenicity tests, randomly selected isolates of the three groups caused root and stem rot on cut-flower and miniature roses. To differentiate AG-CUT and AG-MIN from known AGs of binucleate Rhizoctonia spp., restriction fragment length polymorphism (RFLP) and sequence analyses of a ribosomal (r)DNA internal transcribed spacer (ITS) region were conducted. Among the eight restriction enzymes used, HaeIII produced DNA banding patterns for AG-CUT that differed from those of tester strains and AG-MIN. Additionally, restriction profiles of AG-MIN differed from those of all tester strains. AG-G isolates from cut-flower roses had the same RFLP pattern as the tester strains of AG-G. Based on the results of hyphal anastomosis and RFLP and sequence analysis of an rDNA-ITS region, we propose that AG-CUT be designated AG-T and AG-MIN be designated AG-U, two new AGs of binucleate Rhizoctonia spp. The phylogenetic tree based on the sequence data of the rDNA-ITS region showed that isolates of AG-MIN were in a distinct clade from other AGs, whereas isolates of AG-CUT were in the same clade as those of AG-A. More detailed phylogenetic analysis besides rDNA-ITS region might be necessary for AG classification of binucleate Rhizoctonia spp.