Genome-scale phylogenetic analyses confirm Olpidium as the closest living zoosporic fungus to the non-flagellated, terrestrial fungi

The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium, Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.

Genome-scale phylogenetic analyses confirm Olpidium as the closest living zoosporic fungus to the non-flagellated, terrestrial fungi

The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium, Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.

Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission

ABSTRACT Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus. As with other members of the Tombusvirus genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507–517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008 ). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166–12175, 2013, https://doi.org/10.1128/JVI.01965-13 ) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the β-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the β-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the β-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission. IMPORTANCE Cucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus Olpidium bornovanus. While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166–12175, 2013, https://doi.org/10.1128/JVI.01965-13 ) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507–517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008 ). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the β-annulus at the icosahedral 3-fold axes. Therefore, the β-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.

First Report of Vine Decline of Mature Watermelon Plants Caused by Olpidium bornovanus

In late May 2013, collapse of mature watermelon plants (Citrullus lanatus L.) at first harvest occurred in several drip-irrigated commercial fields in the Coachella Valley, California. Above-ground symptoms consisted of chlorosis, wilting, and death of leaves starting at the crown and progressing rapidly towards the tip of vines. Structural roots of collapsed plants appeared healthy but feeder roots exhibited a brownish discoloration. Microscopic examination revealed that almost all epidermal cells of feeder roots contained either sporangia or resting spores of a fungus tentatively identified, based upon morphological characteristics, as Olpidium bornovanus (Sahtiy.) Karling. No other fungi or fungal-like organisms were microscopically observed in or isolated from structural roots, feeder roots, or vascular tissue of collapsed plants. Leaf, root, and peduncle samples from collapsed plants were tested for Melon necrotic spot virus (MNSV), a virus known to be transmitted by O. bornovanus, and Squash vein yellowing virus (SqVYV), a whitefly-transmitted ipomovirus known to cause watermelon vine decline (1). No MNSV was detected using previously described methods (3). No SqVYV was detected by testing total RNA from symptomatic plants (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) with reverse transcription-PCR using previously described primers and methods (1,2). Genomic DNA was extracted from zoospores of the fungus which were obtained from a single-sporangial isolate maintained on watermelon seedlings. Analysis of ITS 1 and 2 gene sequences and a subsequent search in NCBI GenBank revealed a 99% identity to nucleotide sequences for O. bornovanus (Accession Nos. AB205215 and AB665758). To confirm Koch's postulates, roots of three 5-day-old watermelon seedlings were inoculated by exposure to zoospores (~1 × 105) in a beaker for 2 min and then transplanted into pots containing vermiculite. Pots were irrigated daily and incubated in a growth chamber (25°C, 12-h photoperiod). Controls consisted of non-inoculated watermelon seedlings. The experiment was repeated twice. Within 15 days of inoculation, all inoculated plants were stunted, and roots of stunted plants were brown and most root epidermal cells were filled with either sporangia or resting spores of O. bornovanus. Within 30 days of inoculation, 40 to 60% of the inoculated plants died in all three experiments. No other microorganisms were microscopically observed in or isolated from necrotic roots. Control plants remained symptomless over the duration of the study. Although O. bornovanus has been reported as a root pathogen of melons in greenhouse conditions (3), this is the first worldwide report of the fungus as a root pathogen of watermelons and its association with a late season vine decline in the field. Near-saturated soil conditions resulting from a daily irrigation regime during the latter part of the growing season apparently favored extensive root colonization by this indigenous and opportunistic zoosporic fungus, suggesting that growers should exercise care regarding the duration and frequency of irrigation events. References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) S. Adkins et al. Plant Dis. 1119, 2008. (3) M. E. Stanghellini et al. Plant Dis. 94:163, 2010.

Olpidium bornovanus-Mediated Germination of Ascospores of Monosporascus cannonballus: A Host-Specific Rhizosphere Interaction

Monosporascus cannonballus, a host-specific root-infecting ascomycete, is the causal agent of a destructive disease of melon (Cucumis melo L.) known as vine decline. Ascospores germinate only in the rhizosphere of melon plants growing in field soil. However, no germination occurs in the rhizosphere of melon plants if the field soil is heated to temperatures >50°C prior to infestation with ascospores. This observation suggested that germination is mediated by one or more heat-sensitive members of the soil microflora. Although bacteria or actinomycetes were heretofore suspected as the germination-inducing microbes, our data demonstrate that Olpidium bornovanus, an obligate, host-specific, root-infecting zoosporic fungus, is responsible. In four experiments conducted in autoclaved field soil amended with various population densities of culturally produced ascospores, significant ascospore germination was recorded only in the rhizosphere of cantaloupe seedlings colonized by O. bornovanus.