Isolation and Characterization of Endophyte Bacillus velezensis KOF112 from Grapevine Shoot Xylem as Biological Control Agent for Fungal Diseases

As the use of chemical fungicides has raised environmental concerns, biological control agents have attracted interest as an alternative to chemical fungicides for plant-disease control. In this study, we attempted to explore biological control agents for three fungal phytopathogens causing downy mildew, gray mold, and ripe rot in grapevines, which are derived from shoot xylem of grapevines. KOF112, which was isolated from the Japanese indigenous wine grape Vitis sp. cv. Koshu, inhibited mycelial growth of Botrytis cinerea, Colletotrichum gloeosporioides, and Phytophthora infestans. The KOF112-inhibited mycelial tips were swollen or ruptured, suggesting that KOF112 produces antifungal substances. Analysis of the 16S rDNA sequence revealed that KOF112 is a strain of Bacillus velezensis. Comparative genome analysis indicated significant differences in the synthesis of non-ribosomal synthesized antimicrobial peptides and polyketides between KOF112 and the antagonistic B. velezensis FZB42. KOF112 showed biocontrol activities against gray mold caused by B. cinerea, anthracnose by C. gloeosporioides, and downy mildew by Plasmopara viticola. In the KOF112–P. viticola interaction, KOF112 inhibited zoospore release from P. viticola zoosporangia but not zoospore germination. In addition, KOF112 drastically upregulated the expression of genes encoding class IV chitinase and β-1,3-glucanase in grape leaves, suggesting that KOF112 also works as a biotic elicitor in grapevine. Because it is considered that endophytic KOF112 can colonize well in and/or on grapevine, KOF112 may contribute to pest-management strategies in viticulture and potentially reduce the frequency of chemical fungicide application.

PsGRASP, a Golgi Reassembly Stacking Protein in Phytophthora sojae, Is Required for Mycelial Growth, Stress Responses, and Plant Infection

Golgi reassembly stacking proteins (GRASPs) play important roles in Golgi structure formation, ER stress response, and unconventional secretion in eukaryotic cells. However, GRASP functions in oomycetes haven’t been adequately characterized. Here, we report the identification and functional analysis of PsGRASP, a GRASP-encoding gene from the soybean-infecting oomycete Phytophthora sojae. Transcriptional profiling showed that PsGRASP expression is up-regulated at the infection stages. PsGRASP knockout mutants were created using the CRISPR/Cas9 system. These mutants exhibited impaired vegetative growth, zoospore release and virulence. PsGRASP was involved ER stress responses and altered laccase activity. Our work suggests that PsGRASP is crucial for P. sojae development and pathogenicity.

First Report of White Rust Disease Caused by Albugo koreana on Camelina sativa in China

Camelina sativa, an herbaceous annual plant in the family Brassicaceae, is especially well known for its oilseed crop that produce camelina oil (Hovsepyan et al. 2008). In April 2016, white blister rust disease on C. sativa were observed in a cultivated farmland with an incidence of about 60% in Xinyuan County (43°33'39.17"N, 83°14'54.04"E), Xinjiang, China. Symptoms appeared as light-yellow chlorotic spots on the upper surface of the leaves and white blister on the corresponding lower surface. Blister sori were white, oval to ellipsoidal, scattered or coalesce, and 1.8 to 4 mm in diameter. Two representative voucher specimens were deposited in the Mycological Herbarium of Tarim University (HMUT 2527 and HMUT 2528), Aral, China. Sporangiophores hyaline, clavate or cylindrical, straight to slightly curved, (23.7 to) 27.9 to 37.9 (to 42.1) (av. 31) × (7.9 to) 9.6 to 13.7 (to 15.1) (av. 11.4) μm (n = 30), thick-walled on their lower parts, bearing sporangia in chains. Primary sporangia were globose to subglobose, wall equal thickness, and (9.5 to) 10.6 to 13.2 (to 14.3) (av. 11.9) μm in diameter (n = 50). Secondary sporangia were mostly subglobose to ovoid, with a subtruncated base, and (12.1 to) 13.2 to 16.9 (to 18) (av. 15.1) μm × (11 to) 12.1 to 15 (to 16.1) (av. 13.4) μm in size (n = 50). Oogonia were globose to subglobose, (39.7 to) 42.7 to 51.7 (to 54.1) (av. 48.3) μm in diameter (n = 30), irregular. Oospores were globose to subglobose, brown, (34.5 to) 37 to 42.7 (to 45.2) (av. 41.1) μm in diameter (n = 30), 3 to 5 μm wall in thickness, with single warts, 1.5 to 4 × 2 to 3.5 μm (n = 30). The morphological characteristics of specimens were consistent with those of Albugo koreana (Choi et al. 2007). To confirm the identification, genomic DNA were extracted directly from sori on diseased leaves from isolates HMUT 2527 and HMUT 2528, respectively. The internal transcribed spacer (ITS) rDNA and cytochrome oxidase II (cox2) mtDNA were amplified with primers DC6/LR-0 described by Choi et al. (2006) and cox2-F/cox2-R described by Hudspeth et al. (2000), respectively. A BLASTn search revealed that the ITS rDNA sequences (GenBank accession Nos. MW135444 and MW135445) were 99% (838/844 nucleotides)identical to that of A. koreana from Capsella bursa-pastoris (AY929829), and the cox2 sequences (GenBank accession Nos. MW147150 and MW147151) were 100% (567/567 nucleotides) identical to that of A. koreana from C. bursa-pastoris (AY927048). Based on the concatenated ITS and cox2 sequences, Maximum Likelihood and Bayesian analysis showed that pathogen from C. sativa with the reference isolate of A. koreana (ex C. bursa-pastoris) with high bootstrap support values and maximum posterior probability (100 ML BS and 1.00 BPP, respectively). For pathogenicity, sporangia collected from the infected leaves were suspended in sterile water at 4°C for 2 hours to improve zoospore release, and the zoospore suspension obtained from sporangial suspension (1×105 sporangia/ml) was inoculated to the lower surface of six healthy potted plants. Three non-inoculated plants were served as controls. Each plant was kept in a separate plastic humid chamber in a greenhouse with 25°C and 80% humidity for 15 days. Typical symptoms of white rust pustules developed on the inoculated plants were identical to that observed on the originally infected leaves. Control plants remained symptomless.. Based on morphological characteristics, molecular data, as well as pathogenicity tests, the pathogen on C. sativa was identified as Albugo koreana. A. koreana aslo is reported only on C. bursa-pastoris in Korea (Choi et al. 2007; Farr and Rossman 2020). To our knowledge, this is the first record of white rust disease caused by A. koreana on C. sativa, and the species is new to China. This report represents a new host plant association and a new geographical expansion for this species, presenting a potential threat to camelina production in northwest China.

Aestipascuomyces dupliciliberans gen. nov, sp. nov., the First Cultured Representative of the Uncultured SK4 Clade from Aoudad Sheep and Alpaca

We report on the isolation of the previously-uncultured Neocallimastigomycota SK4 lineage, by two independent research groups, from a wild aoudad sheep rumen sample (Texas, USA) and an alpaca fecal sample (Baden-Württemberg, Germany). Isolates from both locations showed near-identical morphological and microscopic features, forming medium-sized (2–5 mm) white filamentous colonies with a white center of sporangia, on agar roll tubes and a heavy biofilm in liquid media. Microscopic analysis revealed monocentric thalli, and spherical polyflagellated zoospores with 7–20 flagella. Zoospore release occurred through an apical pore as well as by sporangial wall rupturing, a duality that is unique amongst described anaerobic gut fungal strains. Isolates were capable of growing on a wide range of mono-, oligo-, and polysaccharide substrates as the sole carbon source. Phylogenetic assessment based on the D1–D2 28S large rRNA gene subunit (D1–D2 LSU) and internal transcribed spacer-1 (ITS-1) regions demonstrated high sequence identity (minimum identity of 99.07% and 96.96%, respectively) between all isolates; but low sequence identity (92.4% and 86.7%, respectively) to their closest cultured relatives. D1–D2 LSU phylogenetic trees grouped the isolates as a new monophyletic clade within the Orpinomyces–Neocallimastix–Pecoramyces–Feramyces–Ghazallamyces supragenus group. D1–D2 LSU and ITS-1 sequences recovered from the obtained isolates were either identical or displayed extremely high sequence similarity to sequences recovered from the same aoudad sheep sample on which isolation was conducted, as well as several sequences recovered from domestic sheep and few other herbivores. Interestingly, members of the SK4 clade seem to be encountered preferably in animals grazing on summer pasture. We hence propose accommodating these novel isolates in a new genus, Aestipascuomyces (derived from the Latin word for “summer pasture”), and a new species, A. dupliciliberans. The type strain is Aestipascuomycesdupliciliberans strain R4.

Aestipascuomyces dupliciliberatus gen. nov, sp. nov., the First Cultured Representative of the Uncultured SK4 Clade from Aoudad Sheep and Alpaca

We report on the isolation of the previously uncultured Neocallimastigomycota SK4 lineage by two independent research groups from a wild aoudad sheep rumen sample (Texas, USA) and an alpaca fecal sample (Baden-Württemberg, Germany). Isolates from both locations showed near identical morphological and microscopic features, forming medium-sized white filamentous colonies with a white center of sporangia on agar roll tubes and a heavy biofilm in liquid media. Microscopic analysis revealed monocentric thalli, and spherical polyflagellated zoospores with 7–20 flagella. Zoospore release occurred through an apical pore as well as by sporangial wall rupturing, a duality that is unique amongst described AGF strains. Isolates were capable of growing on a wide range of mono-, oligo-, and polysaccharides substrates. Phylogenetic assessment based on the D1-D2 large rRNA subunit (D1-D2 LSU) and internal transcribed spacer-1 (ITS-1) regions demonstrated high sequence identity (minimum identity of 99.07% and 96.96%, respectively) between all isolates; but low sequence identity (92.4% and 86.7%, respectively) to their closest cultured relatives. D1-D2 LSU phylogenetic trees grouped the isolates as a new monophyletic clade within the Orpinomyces-Neocallimastix-Pecoramyces-Feramyces-Ghazallamyces supragenus group. D1-D2 LSU and ITS-1 sequences from the obtained isolates were either identical, or displayed extremely high sequence similarity to sequences recovered from the same Aoudad sheep sample on which isolation was conducted, as well as several sequences recovered from domestic sheep and few other herbivores. Interestingly, members of the SK4 clade seem to be encountered in animals grazing on summer pasture. We hence propose accommodating these novel isolates in a new genus, Aestipascuomyces (derived from the Latin word for “summer pasture”), and a new species, A. dupliciliberatus. The type strain is Aestipascuomyces dupliciliberatus strain R4.

Transcriptomic and Ultrastructural Signatures of K+-Induced Aggregation in Phytophthora parasitica Zoospores

Most pathogenic oomycetes of the genus Phytophthora spread in water films as flagellated zoospores. Zoospores perceive and produce signals attracting other zoospores, resulting in autoaggregation in vitro or biofilm formation on plant surface. The mechanisms underlying intercellular communication and consequent attraction, adhesion and aggregation are largely unknown. In Phytophthora parasitica, the perception of a K+ gradient induces coordinated motion and aggregation. To define cellular and molecular events associated with oomycete aggregation, we combined transcriptomic and ultrastructural analyses. Results indicate involvement of electroception in K+ sensing. They establish that the transcriptome repertoire required for swimming and aggregation is already fully functional at zoospore release. At the time points analyzed, aggregates are mainly constituted of zoospores. They produce vesicular and fibrillary material discharged at cell-to-cell contacts. Consistently, the signature of transcriptome dynamics during transition to aggregates is an upregulation of genes potentially related to vesicular trafficking. Moreover, transcriptomic and functional analyses show a strong enhancement of carbonic anhydrase activity, indicating that pH homeostasis may contribute to aggregation by acting on both zoospore movement and adhesion. This study poses the molecular and cellular bases of aggregative behavior within oomycetes and expands the current knowledge of ion perception-mediated dissemination of propagules in the rhizosphere.

Phytophthora infestans Sporangia Produced in Artificial Media and Plant Lesions Have Subtly Divergent Transcription Profiles but Equivalent Infection Potential and Aggressiveness

Sporangia of the potato late blight agent Phytophthora infestans are often used in studies of pathogen biology and plant responses to infection. Investigations of spore biology can be challenging in oomycetes because their sporangia are physiologically active and change in response to environmental factors and aging. Whether sporangia from artificial media and plant lesions are functionally equivalent has been a topic of debate. To address these issues, we compared the transcriptomes and infection ability of sporangia from rye-sucrose media, potato and tomato leaflets, and potato tubers. Small differences were observed between the mRNA profiles of sporangia from all sources, including variation in genes encoding metabolic enzymes, cell-wall-degrading enzymes, and ABC transporters. Small differences in sporangia age also resulted in variation in the transcriptome. Taking care to use sporangia of similar maturity, we observed that those sourced from media or plant lesions had similar rates of zoospore release and cyst germination. There were also no differences in infection rates or aggressiveness on leaflets, based on single-spore inoculation assays. Such results are discordant with those of a recent publication in this journal. Nevertheless, we conclude that sporangia from plant and media cultures are functionally similar and emphasize the importance of using “best practices” in experiments with sporangia to obtain reliable results.

Seven new Neocallimastigomycota genera from fecal samples of wild, zoo-housed, and domesticated herbivores: Description ofGhazallomyces constrictusgen. nov., sp. nov.,Aklioshbomyces papillarumgen. nov., sp. nov.,Agriosomyces longusgen. nov., sp. nov.,Capellomyces foraminisgen. nov., sp. nov. andCapellomyces elongatussp. nov.,Joblinomyces apicalisgen. nov., sp. nov.,Khoyollomyces ramosusgen. nov., sp. nov., andTahromyces munnarensisgen. nov., sp. nov

ABSTRACTWe isolated and characterized sixty-five anaerobic gut fungi (AGF, Neocallimastigomycota) strains from fecal samples of five wild (W), one zoo-housed (Z), and three domesticated (D) herbivores in the US states of Texas (TX) and Oklahoma (OK), Wales (WA), and the Indian states of Kerala (KE) and Haryana (HA). Phylogenetic assessment based on D1-D2 region of the large rRNA subunit (LSU) identified seven distinct lineages, with strains recovered from Axis Deer (W-TX) clustering within theOrpinomyces-Neocallimastix-Pecoramyces-Feramycesclade; Boer Goat-domesticated Goat strains (W-TX, D-KE) clustering within theOontomyces-Anaeromyces-Liebetanzomycesclade; and domesticated Goat and Sheep strains (D-HA) as well as Nilgiri Tahr strains (W-KE) forming two distinct clades associated with genusBuwchfawromyces. The remaining three lineages, represented by strains recovered from Mouflon-Boer Goat (W-TX), White Tailed Deer (W-OK), and Zebra-Horse (Z-OK, and D-WA), displayed no specific suprageneric affiliation. All strains displayed monocentric thalli and produced mono/uniflagellate zoospores with the exception of Axis Deer strains, which produced polyflagellate zoospores. Isolates displayed multiple interesting microscopic features including sporangia with tightly constricted necks and fine septa at the base (Axis Deer), papillated and pseudo-intercalary sporangia (White-Tailed Deer), swollen sporangiophores and zoospores with long flagella (Mouflon-Boer Goat), zoospore release through an apical pore followed by either sporangial wall collapse (Axis Deer and Boer Goat-domesticated Goat) or sporangial wall remaining intact after discharge (Zebra-Horse), multi-sporangiated thalli with branched sporangiophores (Zebra-Horse), and short sporangiophores with subsporangial swellings (Nilgiri Tahr). Internal transcribed spacer-1 region (ITS-1) sequence analysis indicated that Zebra-Horse strains are representatives of the AL1 lineage, frequently encountered in culture-independent surveys of the alimentary tract and fecal samples from hindgut fermenters. The other six lineages, five of which were isolated from wild herbivores, have not been previously encountered in such surveys. Our results significantly expand the genus level diversity within the Neocallimastigomycota, and strongly suggest that wild herbivores represent a yet-untapped reservoir of AGF diversity. We propose the creation of seven novel genera and eight novel Neocallimastigomycota species to accommodate these strains, for which we propose the namesAgriosomyces longus(Mouflon and wild Boer Goat),Aklioshbomyces papillarum(White tailed Deer),Capellomyces foraminis(wild Boar Goat) andC. elongatus(domesticated Goat),Ghazallomyces constrictus(Axis Deer),Joblinomyces apicalis(domesticated Goat and Sheep),Khoyollomyces ramosus(Zebra-Horse), andTahromyces munnarensis(Nilgiri Tahr). The type species are strains Axs-31, WT-2, MS-4, BGB-11, GFKJa1916, GFH683, ZS-33, and TDFKJa193, respectively.