Sensitivity of Globisporangium ultimum to the fungicide metalaxyl is enhanced by the infection with a toti-like mycovirus.

In recent years, numerous oomycete mycoviruses have been discovered; however, very few studies have focused on their effects on the host oomycete phenotype. In this study, we investigated the impact of toti-like Pythium ultimum RNA virus 2 (PuRV2) infection on the phytopathogenic soil-borne oomycete Globisporangium ultimum, which serves as a model species for Globisporangium and Pythium, specifically the UOP226 isolate in Japan. We generated a PuRV2-free isogenic line through hyphal tip isolation using high-temperature culture and subsequently compared the phenotypic characteristics and gene expression profiles of UOP226 and the PuRV2-free isogenic line. Our findings revealed that the metalaxyl sensitivity of UOP226 was greater than that of the PuRV2-free isogenic line, whereas the mycelial growth rate and colony morphology remained unchanged in the absence of the fungicide. Furthermore, transcriptome analyses using RNA-seq revealed significant downregulation of ABC-type transporter genes, which are involved in fungicide sensitivity, in UOP226. Our results suggest that PuRV2 infection influences the ecology of G. ultimum in agricultural ecosystems where metalaxyl is applied.

An ecological network approach for detecting and validating influential organisms for rice growth.

How to achieve sustainable food production while reducing environmental impacts is a major concern in agricultural science, and advanced breeding techniques are promising for achieving such goals. However, rice is usually grown under field conditions and influenced by surrounding ecological community members. How ecological communities influence the rice performance in the field has been underexplored despite the potential of ecological communities to establish an environment-friendly agricultural system. In the present study, we demonstrate an ecological-network-based approach to detect potentially influential, previously overlooked organisms for rice (Oryza sativa). First, we established small experimental rice plots, and measured rice growth and monitored ecological community dynamics intensively and extensively using quantitative environmental DNA metabarcoding in 2017 in Japan. We detected more than 1000 species (including microbes and macrobes such as insects) in the rice plots, and nonlinear time series analysis detected 52 potentially influential organisms with lower-level taxonomic information. The results of the time series analysis were validated under field conditions in 2019 by field manipulation experiments. In 2019, we focused on two species, Globisporangium nunn and Chironomus kiiensis, whose abundance was manipulated in artificial rice plots. The responses of rice, namely, the growth rate and gene expression patterns, were measured before and after the manipulation. We confirmed that, especially in the G. nunn-added treatment, rice growth rate and gene expression pattern were changed. In the present study, we demonstrated that intensive monitoring of an agricultural system and the application of nonlinear time series analysis were helpful to identify influential organisms under field conditions. Although the effects of the manipulations were relatively small, the research framework presented here has future potential to harness the ecological complexity and utilize it in agriculture. Our proof-of-concept study would be an important basis for the further development of field-basis system management.

Taxonomic revision of the Typhula ishikariensis complex.

Typhula ishikariensis and the related fungi were separated into three biological species by morphological and physiological characteristics, as well as DNA sequences and mating reactions. We propose that the T. ishikariensis complex should be divided into three species (T. ishikariensis, T. canadensis and T. hyperborea) and two varieties (T. ishikariensis var. ishikariensis and var. idahoensis). Typhula hyperborea was reappraised to be recognized also as a separate species of the T. ishikariensis complex.

Novel Fusari- and Toti-like Viruses, with Probable Different Origins, in the Plant Pathogenic Oomycete Globisporangiumultimum.

To further classify the oomycete viruses that have been discovered in recent years, we investigated virus infection in the plant-parasitic oomycete Globisporangium ultimum in Japan. Double-stranded RNA detection, high-throughput sequencing, and RT-PCR revealed that the G. ultimum isolate UOP226 contained two viruses related to fusarivirus and totivirus, named Pythium ultimum RNA virus 1 (PuRV1) and Pythium ultimum RNA virus 2 (PuRV2), respectively. Phylogenetic analysis of the deduced amino acid sequence of the RNA-dependent RNA polymerase (RdRp) showed that fusari-like PuRV1 belonged to a different phylogenetic group than Plasmopara viticola lesion-associated fusari virus (PvlaFV) 1-3 from oomycete Plasmopara viticola. Codon usage bias of the PuRV1 RdRp gene was more similar to those of fungi than Globisporangium and Phytophthora, suggesting that the PuRV1 ancestor horizontally transmitted to G. ultimum ancestor from fungi. Phylogenetic analysis of the deduced amino acid sequence of the RdRp of toti-like PuRV2 showed a monophyletic group with the other toti-like oomycete viruses from Globisporangium, Phytophthora, and Pl. viticola. However, the nucleotide sequences of toti-like oomycete viruses were not so homologous, suggesting the possibility of convergent evolution of toti-like oomycete viruses.

Identification and Isolation Pattern of Globisporangium spp. from a Sanionia Moss Colony in Ny-Ålesund, Spitsbergen Is., Norway from 2006 to 2018.

Globisporangium spp. are soil-inhabiting oomycetes distributed worldwide, including in polar regions. Some species of the genus are known as important plant pathogens. This study aimed to clarify the species construction of Globisporangium spp. and their long-term isolation pattern in Sanionia moss in Ny-Ålesund, Spitsbergen Is., Norway. Globisporangium spp. were isolated at two-year intervals between 2006 and 2018 at a Sanionia moss colony, Ny-Ålesund, Spitsbergen Is., Norway. The isolates were obtained by using three agar media and were identified based on sequences of the rDNA-ITS region and cultural characteristics. Most of the Globisporangium isolates obtained during the survey were identified into six species. All six species were grown at 0 °C on an agar plate and used to infect Sanionia moss at 4 and/or 10 °C under an in vitro inoculation test. The total isolation frequency of Globisporangium gradually decreased throughout the survey period. The isolation frequency varied among the six species, and four of the species that showed a high frequency in 2006 were rarely isolated after 2016. The results suggested that Globisporangium inhabiting Sanionia moss in Ny-Ålesund has a unique composition of species and that most of the species reduced their population over the recent decade.

First Report of Pythium Cluster B2a Species Causing Root Rot in Welsh Onion.

Hiroshima Prefecture has the highest production area of hydroponically grown Welsh onions (Allium fistulosum L.) in Japan. Since the cultivation began in 1988, root rot (Fig. 1A) followed by leaf browning (Fig. 1B) has caused significant economic losses. Approximately 80% (loss of 45 million JPY) of plant loss occurred from May to Sep 2009 (Shimizu, unpublished), and the disease was observed again in 2020. Diseased Welsh onions (five to six leaf stage) were collected in 2009. Abundant nonseptate hyphae of Pythium-like organisms were observed in the rotted roots (Fig. 1C). Disinfected symptomatic tissue samples were placed on NARF medium (Morita and Tojo 2007) and incubated at 25°C for 3 to 7 days. Six Pythium-like organisms were isolated, and their morphological features on a grass blade culture, potato carrot agar (PCA) (van der Plaats-Niterink 1981), cornmeal agar (CMA) and V8 juice agar (Miller 1955) were examined. Hyphal growth rates from 1-46°C were measured by culturing on PCA. The ribosomal internal transcribed spacer (ITS) regions and mitochondrial COI of the isolates were amplified and sequenced according to Ueta and Tojo (2016). All six isolates obtained showed similar morphology, hyphal growth rates, and sequences of ITS and COI. Detailed descriptions are provided here for the representative isolate 72 (MAFF246451). The isolate produced asexual structures but did not form sexual structures, including oogonia, antheridia, and oospores on all the media used. Hyphae were up to 6.8 µm wide. Appressoria were knob-like terminations (Fig. 1D). Sporangia were filamentous and indistinguishable from the hyphae. Zoospores (Fig. 1E) were formed at 5-25°C. The diameter of encysted zoospores ranged 7.4-10.1 (av. 8.9) µm (Fig. 1F). Cardinal temperatures for hyphal growth on PCA were 5°C min, 28-31°C opt, and 35°C max. The daily growth rate at 25°C was 15.0 mm per day. The sequence analysis of all isolates, including isolate 72 (GenBank ac nos AB700596 for ITS, LC630955 for COI) showed the present isolates belonged to Pythium Cluster B2a (Robideau et al. 2011) (Fig. 2). Based on these features, the six isolates were identified as Pythium Cluster B2a sp. In the inoculation test, isolate 72 was cultured on CMA at 25°C for 5 days. Mycelium disks (5 mm diam) obtained from the culture were placed on the primary roots of 8-day-old Welsh onion seedlings (cv Koutou), which were grown at a density of six plants on rock wool cubes moistened with tap water in a 50 mL plastic pot. The inoculated and non-inoculated plants were grown at 28°C for 7 days in a growth chamber. The experiment was repeated twice using three pots per replication. The plants inoculated with isolate 72 wilted, and their roots rotted 7 days after inoculation. No disease was found observed on the non-inoculated plants. The isolate of Pythium Cluster B2a sp. was consistently re-isolated from the diseased plants, thus, fulfilling Koch's postulates. Pythium Cluster B2a sp. causing stem rot on lettuce has been recorded in Italy (Garibaldi et al. 2017). To our knowledge, this is the first report of Pythium Cluster B2a sp. on Welsh onions. Since significant losses to root rot of Welsh onion have occurred in Japan, identification of the causal organism will enable the development of management practices to reduces losses.

Chemical genetic approach using ß-rubromycin reveals that a RIO kinase-like protein is involved in morphological development in Phytophthora infestans.

To characterize the molecular mechanisms underlying life-stage transitions in Phytophthora infestans, we initiated a chemical genetics approach by screening for a stage-specific inhibitor of morphological development from microbial culture extracts prepared mostly from actinomycetes from soil in Japan. Of the more than 700 extracts, one consistently inhibited Ph. infestans cyst germination. Purification and identification of the active compound by ESI-MS, 1H-NMR, and 13C-NMR identified ß-rubromycin as the inhibitor of cyst germination (IC50 = 19.8 µg/L); ß-rubromycin did not inhibit growth on rye media, sporangium formation, zoospore release, cyst formation, or appressorium formation in Ph. infestans. Further analyses revealed that ß-rubromycin inhibited the germination of cysts and oospores in Pythium aphanidermatum. A chemical genetic approach revealed that ß-rubromycin stimulated the expression of RIO kinase-like gene (PITG_04584) by 60-fold in Ph. infestans. Genetic analyses revealed that PITG_04584, which lacks close non-oomycete relatives, was involved in zoosporogenesis, cyst germination, and appressorium formation in Ph. infestans. These data imply that further functional analyses of PITG_04584 may contribute to new methods to suppress diseases caused by oomycetes.

A novel toti-like virus from a plant pathogenic oomycete Globisporangium splendens.

We investigated virus infection in the plant pathogenic oomycete Globisporangium splendens, formerly classified as Pythium splendens, in Japan. From 12 strains investigated, three strains contained virus-like double-stranded (dsRNA). Next-generation sequencing revealed that the G. splendens strain MAFF 425508 and MAFF 305867 contained a virus related to toti-like viruses, that we named Pythium splendens RNA virus 1 (PsRV1). PsRV1 has a ca. 5700 nt-length genome encoding two overlapping open reading frames (ORFs). The ORF2 encodes an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis with deduced RdRp amino acid sequences indicated that PsRV1 was closely related to Pythium polare RNA virus 1 (PpRV1) from G. polare infecting mosses in the Arctic. PsRV1 was vertically transmitted through the hyphal swellings, vegetative organs of G. splendens, in a temperature-dependent manner. Also, we showed that PsRV1 infected in a symptomless manner.

Antimicrobial agent isolated from Coptidis rhizome extract incubated with Rhodococcus sp. strain BD7100.

Coptidis rhizome (CR) is a widely used herbal medicine that contains protoberberine-type alkaloids. CR extract exhibits various pharmacologic activities. A previous study reported the isolation of Rhodococcus sp. strain BD7100 as a berberine (BBR)-utilizing bacterium, and the BBR-degradation pathway has been investigated. When we incubated strain BD7100 cells with CR extract, the number of viable cells declined with the degradation of components in the CR extract, and the culture broth exhibited antibacterial activity against strain BD7100. These results suggest that CR extract cultured in the presence of strain BD7100 contains one or more antibacterial agents. In this study, we isolated coptirhoquinone A (1) from CR extract incubated with strain BD7100 in Luria-Bertani (LB) medium, and the structure was elucidated using NMR and MS analysis. We also report the total synthesis and antimicrobial activities of 1 against bacteria, fungi, and Pythium sp.

A novel non-segmented double-stranded RNA virus from an Arctic isolate of Pythium polare.

We investigated virus infection in the oomycete Pythium polare from the Arctic. From 39 isolates investigated, 14 contained virus-like double-stranded RNA (dsRNA). Next generation sequencing revealed that the P. polare isolate OPU1176 contained three different virus-like sequences. We determined the full-length genome sequence of one of them. The 5397 nt-length genome had two overlapped open reading frames (ORFs) consistent with a toti and toti-like viruses, that we named Pythium polare RNA virus 1 (PpRV1). The ORF2 encoded an RNA-dependent RNA polymerase (RdRp). The shifty heptamer motif and RNA pseudoknot were predicted near the stop codon of ORF1, implying that the RdRp could be translated as a fusion protein with the ORF1 protein. Phylogenetic analysis with deduced RdRp amino acid sequences indicated that oomycete virus PpRV1 was closely related to the unclassified arthropod toti-like viruses. The comparison of PpRV1-free and -infected lines suggested that PpRV1 infected in a symptomless manner.

Genome sequence of a novel partitivirus identified from the oomycete Pythium nunn.

The mycoparasitic oomycete Pythium nunn isolate UZ415 contains two double-stranded RNAs (dsRNAs) of different sizes. The 1707-nt dsRNA1 and the 1475-nt dsRNA2 potentially encode an RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively, with sequence similarity to the RdRp and CP of gammapartitiviruses (< 57% and < 36%). Phylogenetic analysis of the deduced RdRp amino acid sequences indicated that the virus identified from P. nunn is classifiable as a distinct member of the genus Ganmmapartitivirus in the family Partitiviridae. This virus isolate is hereby named Pythium nunn virus 1 (PnV1).

The effect of tar spot pathogen on host plant carbon balance and its possible consequences on a tundra ecosystem.

In Arctic tundra, plant pathogens have substantial effects on the growth and survival of hosts, and impacts on the carbon balance at the scale of ecological systems. To understand these effects on carbon dynamics across different scales including plant organ, individual, population and ecosystem, we focused on two primary factors: host productivity reduction and carbon consumption by the pathogen. We measured the effect of the pathogen on photosynthetic and respiratory activity in the host. We also measured respiration and the amount of carbon in the pathogen. We constructed a model based on these two factors, and calculated pathogenic effects on the carbon balance at different organismal and ecological scales. We found that carbon was reduced in infected leaves by 118% compared with healthy leaves; the major factor causing this loss was pathogenic carbon consumption. The carbon balance at the population and ecosystem levels decreased by 35% and 20%, respectively, at an infection rate of 30%. This case study provides the first evidence that a host plant can lose more carbon through pathogenic carbon consumption than through a reduction in productivity. Such a pathogenic effect could greatly change ecosystem carbon cycling without decreasing annual productivity.

Effects of vermicompost water extract prepared from bamboo and kudzu against Meloidogyne incognita and Rotylenchulus reniformis.

A series of experiments in laboratory, greenhouse, and field were conducted to compare the nematode suppressive effect of vermicompost tea (VCT) prepared from vermicompost with moso-bamboo (Phyllostachys edulis (Carrière) J. Houz.) and kudzu (Pueraria lobata (Willd) Ohwi) as feed stock (weed VCT) to that prepared from vegetable food waste (vegetable VCT) against Meloidogyne incognita and Rotylenchulus reniformis. Two laboratory trials were conducted by incubating eggs of M. incognita and R. reniformis in weed VCT or vegetable VCT over 1 wk. These trials revealed that although both VCTs suppressed M. incognita egg hatching compared to water control, only weed VCT suppressed R. reniformis egg hatching. In addition, both VCTs suppressed the mobility of second stage juveniles (J2s) of M. incognita equally compared to water control though suppression from weed VCT performed inconsistently between the trials. When root penetration of M. incognita on cucumber drenched with VCT on one side of a split-root system in a greenhouse sterile sand-soil mix was examined, weed VCT suppressed root penetration of M. incognita on the other side of the root in two trials, but vegetable VCT was only effective in one trial. However, both VCTs did not suppress R. reniformis root penetration. When the effect of the VCTs was examined in two cowpea (Vigna unguiculata) field trials, drenching of VCTs did not affect cowpea growth and yield, but weed VCT reduced root-gall index compared to the water control in both trials. Although both VCTs did not reduce the number of M. incognita and R. reniformis in soil, weed VCT did increase omnivorous nematodes in the second trial, indicating a gradual improvement of soil food web structure through VCT drenching over time. Overall, performance of weed VCT was more consistent than vegetable VCT for plant-parasitic nematodes suppression.A series of experiments in laboratory, greenhouse, and field were conducted to compare the nematode suppressive effect of vermicompost tea (VCT) prepared from vermicompost with moso-bamboo (Phyllostachys edulis (Carrière) J. Houz.) and kudzu (Pueraria lobata (Willd) Ohwi) as feed stock (weed VCT) to that prepared from vegetable food waste (vegetable VCT) against Meloidogyne incognita and Rotylenchulus reniformis. Two laboratory trials were conducted by incubating eggs of M. incognita and R. reniformis in weed VCT or vegetable VCT over 1 wk. These trials revealed that although both VCTs suppressed M. incognita egg hatching compared to water control, only weed VCT suppressed R. reniformis egg hatching. In addition, both VCTs suppressed the mobility of second stage juveniles (J2s) of M. incognita equally compared to water control though suppression from weed VCT performed inconsistently between the trials. When root penetration of M. incognita on cucumber drenched with VCT on one side of a split-root system in a greenhouse sterile sand-soil mix was examined, weed VCT suppressed root penetration of M. incognita on the other side of the root in two trials, but vegetable VCT was only effective in one trial. However, both VCTs did not suppress R. reniformis root penetration. When the effect of the VCTs was examined in two cowpea (Vigna unguiculata) field trials, drenching of VCTs did not affect cowpea growth and yield, but weed VCT reduced root-gall index compared to the water control in both trials. Although both VCTs did not reduce the number of M. incognita and R. reniformis in soil, weed VCT did increase omnivorous nematodes in the second trial, indicating a gradual improvement of soil food web structure through VCT drenching over time. Overall, performance of weed VCT was more consistent than vegetable VCT for plant-parasitic nematodes suppression.

Typhula cf. subvariabilis, new snow mould in Antarctica.

We collected snow blight of moss, Polytrichum juniperinum on King George Island, maritime Antarctica. Host died in a circle of about 10-30 cm after snow melts. Clamp connected hyphae and no sclerotia were observed on tip of host leaves. DNA sequence of ITS region from moss symptoms were perfectly matched with fruit bodies of Typhula sp. on Macquarie Island in the maritime Antarctica and high homology with Typhula cf. subvariabilis from Iran. Therefore, we suggested that T. cf. subvariabilis caused snow blight on moss in Antarctica, and this is first record of Typhula snow blight in Southern Hemisphere. These results also suggested that fungi in same genera gained similar ecological niche in both Polar Regions.

Globisporangium oryzicola sp. nov., causing poor seedling establishment of directly seeded rice.

A new species, Globisporangium oryzicola, was isolated from directly seeded rice seedlings, and from soils of paddy fields and an uncultivated field. Despite their different origins, five of the seven isolates studied caused poor seedling establishment of rice in a laboratory inoculation experiment. The species is characterized by oogonia with smooth-walled or sometimes one projection, with one to two antheridia, and aplerotic oospores. Hyphal swellings were rarely observed. Phylogenetic analyses based on the internal transcribed spacer region of the ribosomal RNA gene and mitochondrial cytochrome c oxidase subunit 1 and 2 genes confirmed that the species differed from other Globisporangium species. This novel species is described and illustrated in detail.

Isolation of growth inhibitors of the snow rot pathogen Pythium iwayamai from an arctic strain of Trichoderma polysporum.

Growth inhibitors were isolated from an arctic strain of Trichoderma polysporum, and the structures were elucidated and the in vitro inhibitory effects of these compounds against Pythium iwayamai were investigated. Eleven compounds were isolated; four showed a concentration-dependent growth-inhibitory effect against P. iwayamai. None of these compounds have been reported previously as substances with antimicrobial activity against P. iwayamai. One of these four compounds inhibited the growth of the pathogen at 33 µg ml(-1) concentration during a 15-day incubation at 20 °C. This effect was comparable to that of chloroneb (1: 1,4-dichloro-2,5-dimethoxybenzene), a fungicide with activity against P. iwayamai. Thus, the results of the present study show that the arctic strain of T. polysporum can be an effective source of antibiotics with activity against the snow rot pathogen, P. iwayamai.

Pythium kandovanense sp. nov., a fungus-like eukaryotic micro-organism (Stramenopila, Pythiales) isolated from snow-covered ryegrass leaves.

Pythiumkandovanense sp. nov. (ex-type culture CCTU 1813T = OPU 1626T = CBS 139567T) is a novel oomycete species isolated from Lolium perenne with snow rot symptoms in a natural grassland in East-Azarbaijan province, Iran. Phylogenetic analyses based on sequence data from internal transcribed spacer (ITS)-rDNA, coxI and coxII mitochondrial genes clustered our isolates in Pythium group E as a unique, well supported clade. Pythium kandovanense sp. nov. is phylogenetically and morphologically distinct from the other closely related species in this clade, namely Pythium rostratifingens and Pythium rostratum. Pythium kandovanense sp. nov. can be distinguished from these two species by its cylindrical sporangia and lower temperatures for optimum and maximum growth rate. The development of zoospores released through a shorter discharge tube is an additional morphological feature which can be used to differentiate Pythium kandovanense sp. nov. from Pythium rostratifingens. Laboratory inoculation tests demonstrated the pathogenicity of Pythium kandovanense sp. nov. to L. perenne under wet cold (0-3 °C) conditions.

Surviving freezing in plant tissues by oomycetous snow molds.

Oomyceteous snow molds, Pythium species, were reported to be less tolerant to chilling and freezing temperatures than other snow mold taxa. However, Pythium species are often found to be pathogenic on mosses in Polar Regions. We investigated the frost resistance of Pythium species from Temperate (Hokkaido, Japan) and Subantarctic Regions. Free mycelia and hyphal swellings, structures for survival, of Pythium iwayamai and Pythium paddicum lost viability within freeze-thaw 3 cycles; however, mycelia in host plants survived the treatment. It was reported that fungi in permafrost are characterized both by the presence of natural cryoprotectants in these ecotopes and by the ability to utilize their inherent mechanisms of protection. It is conceivable that plant substrates or derivatives thereof are natural cryoprotectants, enabling them to provide advantageous conditions to microorganisms under freezing conditions. Our results are the first to experimentally support this hypothesis.

Pythium polare, a new heterothallic oomycete causing brown discolouration of Sanionia uncinata in the Arctic and Antarctic.

Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis.

Colonization of Pythium oligandrum in the tomato rhizosphere for biological control of bacterial wilt disease analyzed by real-time PCR and confocal laser-scanning microscopy.

It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 x 10(5) oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.