FFGA1 Protein Is Essential for Regulating Vegetative Growth, Cell Wall Integrity, and Protection against Stress in Flammunina filiformis.

Flammulina filiformis is a popular mushroom which has been regarded as a potential model fungus for mycelium growth, fruiting body development, and stress response studies. Based on a genome-wide search, four genes encoding heterotrimeric G protein α subunits were identified in F. filiformis. The data of conserved domain analysis showed that these genes contain only one subgroup I of Gα subunit (Gαi), similar to many other fungi. To explore the function of Gαi, FfGa1 over-expression (OE) and RNA interference (RNAi) strains were generated using the Agrobacterium tumefaciens-mediated transformation (ATMT) approach. RNAi transformant strains showed remarkably reduced growth on PDA medium and added sensitivity to cell wall-enforcing agents with maximum growth inhibition, but showed better growth in response to hypertonic stress-causing agents, while OE strains exhibited more resistance to thermal stress and mycoparasite Trichoderma as compared to the wild-type and RNAi strains. Taken together, our results indicated that FfGa1 positively regulates hyphal extension, and is crucial for the maintenance of cell wall integrity and protection against biotic and abiotic (hypertonic and thermal) stress.

Morphological and molecular features of early regeneration in the marine annelid Ophryotrocha xiamen.

Regeneration capability varies in the phylum Annelida making them an excellent group to investigate the differences between closely related organisms. Several studies have described the process of regeneration, while the underlying molecular mechanism remains unclear, especially during the early stage (wound healing and blastema formation). In this study, the newly identified Ophryotrocha xiamen was used to explore the early regeneration. The detailed morphological and molecular analyses positioned O. xiamen within 'labronica' clade. We analyzed the morphological changes during regeneration process (0-3 days post amputation) and molecular changes during the early regeneration stage (1 day post amputation). Wound healing was achieved within one day and a blastema formed one day later. A total of 243 DEGs were mainly involved in metabolism and signal transduction. Currently known regeneration-related genes were identified in O. xiamen which could help with exploring the functions of genes involved in regeneration processes. According to their conserved motif, we identified 8 different Hox gene fragments and Hox5 and Lox2 were found to be absent in early regeneration and during regular growth. Our data can promote further use of O. xiamen which can be used as an experimental model for resolving crucial problems of developmental biology in marine invertebrates.

First report of powdery mildew on Astragalus sinicus (Chinese milk vetch) caused by Erysiphe trifoliorum in China.

Astragalus sinicus L., (Chinese milk vetch) is a traditional leguminous green manure that plays a significant role in maintaining paddy soil fertility to enhance yield and the quality of rice in China. It is also found in gardens, roadsides, farms, fields, riverbanks, open wastelands, and is often used as livestock feed. From February 2019 to 2021, severe powdery mildew infections were observed on hundreds of A. sinicus grown in gardens and at roadsides of Fuzhou city, China. The disease incidence was up to 100% on leaves and stems of A. sinicus. White superficial fungal colonies (circular to irregular patches) were present on both sides of the leaves. Hyphae were flexuous to straight, branched, 4 to 8 µm in width, and septate. Hyphal appressoria were lobulate and solitary or in opposite pairs. Conidiophores were erect and straight, hyaline, and 60 to 120 × 8 to 10 µm (n=30). Foot cell was cylindrical, straight to slightly curved, 22 to 38 × 8 to 10 µm, followed by two to three shorter cells. Conidia were cylindrical-oval to doliiform, 30 to 48 × 13.5 to 24 µm with a length/width ratio of 1.6 to 2.4 (n = 30), formed singly, and without fibrosin bodies. Conidial germ tubes were produced subterminal position. No chasmothecia were found in the collected samples. The morphological characteristics of asexual structures were consistent with the descriptions of E. trifoliorum (Wallr.) U. Braun in Braun and Cook (2012). To verify the identification of the pathogen, the ITS and the part of large subunit (LSU) rDNA gene of the isolates were amplified using ITS1/ITS4 and LSU1/ LSU2 primers (Scholin et al. 1994 and White et al. 1990, respectively) and sequences were deposited in GenBank (ITS: MZ021332, MZ021333; LSU: MZ021334, MZ021335). In BLASTn searches, the ITS and LSU sequences were 99 to 100% identical with those of E. trifoliorum parasitic on Lathyrus magellanicus (LC010015), Medicago littoralis (LC270860), Melilotus officinalis (LC009924) and Trifolium spp., (MN216308, KY660821), as well as E. baeumleri (Bradshaw et al. 2021) on Vicia nigricans (LC010014). Pathogenicity test was performed by gently pressing a diseased leaf onto 10 young leaves of three healthy potted plants, while three non-inoculated plants were used as controls. All plants were maintained in a greenhouse at 20 to 25°C, without humidity control, and natural light. Symptoms developed 7 days after inoculation, whereas the control leaves remained symptomless. The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Powdery mildew on A. sinicus has been reported as E. pisi and E. polygoni from Korea and China (Shin, 2000; Tai 1979), respectively. Amano (1986) listed E. pisi and Microsphaera astragali (now E. astragali) on A. sinicus from China and Japan. To our knowledge, this is the first report of powdery mildew caused by E. trifoliorum on A. sinicus in China and in general. E. astragali is the most common and widespread powdery mildew species on Astragalus spp. (Braun and Cook 2012) and would be expected on A. sinicus, but this species is genetically clearly different from E. trifoliorum (Bradshaw et al. 2021). The E. trifoliorum complex (clade) is composed of several morphologically well-distinguishable species, besides E. trifoliorum also including E. baeumleri (on Vicia spp.), E. hyperici (on Hypericum spp.), and E. euonymi (on Euonymus spp.), but based on a combination of sequence plus host identity, the collection on A. sinicus can be assigned to E. trifoliorum (Bradshaw et al. 2021). The information in this study extended the host range of E. trifoliorum as well as future studies on A. sinicus in relation to powdery mildew outbreaks in China. References: Amano (Hirata), K. 1986. Host Range and Geographical Distribution of the Powdery Mildew Fungi. Japan Scientific Societies Press, Tokyo, 741 pp. Bradshaw, M., et al. 2021. Mycologia. (In press) Braun, U., Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, the Netherlands. Scholin, C. A., et al. 1994. J. Phycol. 30:999. Shin, H.D. 2000. Erysiphaceae of Korea. National Institute of Agricultural Science and Technology, Suwon, Korea, 320 pp. Tai, F.L. 1979. Sylloge Fungorum Sinicorum. Sci. Press, Acad. Sin., Peking, 1527 pp. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

A New Protocruzia Species (Ciliophora: Protocruziida) Isolated From the Mariana Trench Area.

A new species of Protocruzia, isolated from the deep-sea Pacific Ocean (>3,000-m depth) in the vicinity of the Mariana Trench, is described based on morphological and molecular data. The systematic status of the ciliate genus Protocruzia has long been highly ambiguous, and Protocruzia species have been assigned to an independent class until recently. In the present study, we described Protocruzia marianaensis sp. n. as a small (25-32 × 14-17 µm in vivo) drop-shaped ciliate, with longitudinal furrows along the ciliary rows on the right side, six adoral membranelles, eight somatic kineties, and one macronucleus comprising 7-11 nuclear globules. Phylogenetic analyses inferred from small subunit rRNA gene sequences revealed that seven Protocruzia species in the phylogenetic tree formed a fully supported clade representing an independent class. Protocruzia marianaensis sp. n. was established to be most closely related to Protocruzia adhaerens, with a sequence similarity of 96.64%, and was found to be able to survive at both atmospheric pressure and hydrostatic pressure of 320 bar, thereby indicating effective barotolerance.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Sigesbeckia orientalis in China.

Sigesbeckia orientalis L., (St Paul's wort) is an annually grown natural herb of Asteraceae with a long therapeutic history for a wide range of inflammation-related diseases in China (Zhong et al. 2019). In June 2020, typical symptoms of powdery mildew were observed on 30% of wild S. orientalis plants grown along the roadsides and gardens in Minjiang University, Fuzhou, China. Circular to irregular white powdery fungal colonies were observed on both surfaces of the leaves and young stems, causing necrosis and premature senescence. Fungal hyphae were epigenous, flexuous to straight, branched, and septate. Appressoria on the hyphae were nipple-shaped or nearly absent. Conidiophores were straight, 30 to 210× 8 to 12 µm, and produced 3 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindrical, 45 to 75 ×10 to 12 µm, followed by 1 to 2 shorter cells. Conidia were hyaline, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 18 to 23 µm with distinct fibrosin bodies. Germ tubes were produced from a lateral position on the conidia. Chasmothecia were not observed on the infected leaves. Based on anamorph characteristics, fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). For molecular identification, total genomic DNA was extracted (Mukhtar et al. 2018) from fungal colonies on infected leaves of five collections separately. For each DNA sample, the part of LSU and ITS regions were amplified using primers LSU1/LSU2 and ITS1/ITS4 (Scholin et al. 1994; White et al. 1990), respectively. A BLAST search revealed 100 % sequences similarity with P. xanthii sequences reported on Ageratum conyzoides (KY274485), Eclipta prostrata (MT260063), Euphorbia hirta (KY388505), Sonchus asper (MN134013), and Verbena bonariensis (AB462804). Representative sequences (ITS: MZ613309; LSU: MZ614707) of an isolate were deposited in GenBank. The phylogenetic analysis also grouped the obtain sequences into P. xanthii clade. Pathogenicity was confirmed by gently pressing the infected leaves onto young leaves of five healthy one-month-old S. orientalis plants, while three non-inoculated plants were used as controls. All plants were maintained in a greenhouse at 25 ± 2°C. After, seven days, white powdery colonies were observed on inoculated plants, whereas controls remained mildew-free. On inoculated leaves, the fungus was morphologically and molecularly identical to the fungus on the original specimens. P. xanthii has been reported as a significant damaging pathogen on a wide range of plants in China (Farr and Rossman 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. orientalis in China as well as worldwide. S. orientalis is one of the most important commercial Chinese medicinal herbs and the occurrence of powdery mildew is a threat to its production, quality, and marketability. References: Braun, U., and Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 9 October 2021. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Zhong, Z., et al., 2019. Chin. Med. (U. K.) 14, 1-12. 10.1186/s13020-019-0260-y.

The Fvclp1 gene regulates mycelial growth and fruiting body development in edible mushroom Flammulina velutipes.

Fruiting body development in Agaricomycetes represents the most complex and unclear process in the fungi. Mating type pathways (A and B) and transcription factors are important regulators in the sexual development of mushrooms. It is known that clampless1 (clp1) is an additional gene that participate under the homeodomain (HD) genes in the matA pathway and clp1 inactivation blocks clamps formation in Coprinopsis cinerea. In this study we identified and analyzed a homologous Fvclp1 gene in the edible mushroom Flammulina velutipes. The coding sequence of the Fvclp1 was 1011 bp without intron interruption, encoding a protein of 336 amino acids. To exhibit the role of Fvclp1 in clamp development and fruiting body formation, knockdown and overexpression mutants were prepared. No significant difference was observed in the monokaryotic hyphal morphology of overexpression and knockdown transformants. In the dikaryotic hyphae from the compatible crossings between the wild-type L22 strain and Fvclp1 knockdown or overexpression mutants, clamp connections developed. However, knockdown mutants could generate fewer fruiting bodies than the wild-type strain. On the contrary, reduced mycelial growth rate but improved fruiting ability was observed in the dikaryotic Fvclp1 overexpression mutants as compared to the wild-type strain. These results indicate that Fvclp1 is necessary and actively involved in fruiting body development in F. velutipes. Overall, these findings suggest that further studies on the function of Fvclp1 would advance our understanding of sexual reproduction and fruiting body development in edible mushrooms.

Synergistically promoting plant health by harnessing synthetic microbial communities and prebiotics.

Soil-borne diseases cause serious economic losses in agriculture. Managing diseases with microbial preparations is an excellent approach to soil-borne disease prevention. However, microbial preparations often exhibit unstable effects, limiting their large-scale application. This review introduces and summarizes disease-suppressive soils, the relationship between carbon sources and the microbial community, and the application of human microbial preparation concepts to plant microbial preparations. We also propose an innovative synthetic microbial community assembly strategy with synergistic prebiotics to promote healthy plant growth and resistance to disease. In this review, a new approach is proposed to improve traditional microbial preparations; provide a better understanding of the relationships among carbon sources, beneficial microorganisms, and plants; and lay a theoretical foundation for developing new microbial preparations.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Bidens pilosa in China.

Bidens pilosa L., (spanish needle), is a wild, flowering plant of Asteraceae, that is grown in gardens, fields, roadsides, and riverbanks in Fuzhou, China. It is also used in traditional folk medicine for a broad range of ailments in China. In March 2019 and 2020, hundreds of B. pilosa growing along the roadsides, and gardens in the districts of Minhou and Jinshan were observed to be severely affected by a powdery mildew with approximately 80% disease incidence. Symptoms appeared as circular to irregular small white, powdery patches, typically on the adaxial sides of leaves and progressed to coalescent colonies on the leaves. As the disease developed, the infected leaves became wilted and senesced. Mycelia on leaves were superficial and solitary appressoria were slightly to distinctly nipple-shaped. Conidiophores were erect, 120 to 230 × 10 to 12 µm, and produced two to five conidia in chains with a sinuate outline. Foot-cells were erect, cylindrical, and 60 to 110 µm long. Conidia were hyaline, ellipsoid to barrel-shaped, 26 to 40 × 18 to 24 µm, and devoid of distinct fibrosin bodies. Germ tubes were long and produced at the perihilar position of the conidia. No chasmothecia were observed. Morphological characteristics overlapped with Golovinomyces ambrosiae, G. cichoracearum, and G. spadiceus (Braun and Cook 2012) on hosts within the Asteraceae tribe Heliantheae (Takamatsu et al. 2013). For molecular identification, ITS and IGS regions as well as partial LSU of two representative collections (MJU-IM019- MJU-IM020), were amplified using ITS1/ITS4, IGS-12a/ NS1R and LSU1/LSU2 primers (Carbone & Kohn, 1999; Scholin et al. 1994; White et al. 1990), respectively. The resulting sequences were deposited in GenBank (ITS: MW965777, MW965778; LSU: MW965787, MW965788; IGS: MW981256, MW981257). A BLAST search revealed 99 to 100 % sequence similarity to G. ambrosiae sequences (KX987303, AB769421, AB077689, AB769426, AB077643, and AB769425). Phylogenetic analysis of ITS, LSU and IGS also grouped obtained sequences within the G. ambrosiae complex (Qiu et al. 2020). Pathogenicity was confirmed through inoculation by gently pressing infected leaves onto leaves of five healthy, potted, young B. pilosa plants, while five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25 ± 2°C. Inoculated plants developed symptoms after 7 to 10 days, whereas the control plants remained symptomless. The morphology of the resulting fungus on inoculated plants was identical to that originally observed on diseased plants. Podosphaera spp., have been reported on B. pilosa (Farr & Rossman 2021) from North America, Africa, and Asia. To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on B. pilosa in China and Asia. Wild populations of B. pilosa may be the primary source of powdery mildew inoculum for commercial Asteraceae members and may warrant consideration in the control of this disease. References: Braun, U., and Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, The Netherlands. Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 18 April 2021. Qiu, P. L., et al. 2020. BMC Microbiol. 20:1. Scholin, C. A., et al. 1994. J. Phycol. 30:999. Takamatsu, S., et al. 2013. Mycologia 105:1135. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

First Report of Postharvest Blue Mold decay caused by Penicillium expansum on Lemon (Citrus limon) fruit in China.

Lemon (Citrus limon) is one of the most important commercial (both dried and fresh) citrus fruits in China. In the spring of 2019, postharvest blue mold decay was observed at an incidence of 3-5% on lemon fruit at the local markets in Beijing, China. Fruit lesions were circular, brown, soft, and watery, and rapidly expanded at 25°C. To isolate the causal organism, small pieces (2 mm3) were cut from the lesions, surface-sterilized for 1 min in 1.5% NaOCl, rinsed three times with sterilized water, dried with sterile filter paper, placed onto potato dextrose agar (PDA) medium, and incubated at 25°C for 6 days. Eight morphologically similar single-colony fungal isolates were recovered from six lemon fruit. Colony surfaces were bluish-green on the upper surface and cream to yellow-brown one the reverse. Hyphae on colony margins were entirely subsurface and cream in color. Mycelium was highly branched, septate, and colorless, and conidiophores were 250 to 450 × 3.0 to 4.0 µm in size. Stipe of conidiophores were smooth-walled, bearing terminal penicilli, typically terverticillate or less commonly birverticillate, rami occurring singly, 16 to 23 × 3.0 to 4.0 µm, metulae in 3 to 6, measuring 12 to 15 × 3.0 to 4.0 µm. Phialides were ampulliform to almost cylindrical, in verticils of 5 to 8, measuring 8 to 11 × 2.5 to 3.2 µm with collula. Conidia were smooth-walled, ellipsoidal, measuring 3.0 to 3.5 × 2.5 to 3.0 µm. According to morphological characteristics, the fungus was identified as Penicillium expansum (Visagie et al. 2014). For molecular identification, genomic DNA of eight fungal isolates was extracted, regions of the beta-tubulin (TUB), and calmodulin (CAL) genes and ITS region, were amplified using Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 primers respectively. Obtained sequences of all isolates were identical to sequences of the representative isolate YC-IK12, which was submitted in the GenBank. BLAST results of YC-IK12 sequences (ITS; MT856700: TUB; MT856958: CAL; MT856959) showed 98 to 100% similarity with P. expansum accessions (NR-077154, LN896428, JX141581). For pathogenicity tests, 10 µl of conidial suspension (10 × 105 conidia/ml) from seven-day-old YC-IK12 culture was inoculated using a sterilized needle into the surface of each five asymptomatic disinfected lemons. As a control, three lemons were inoculated using sterile distilled water. All inoculated lemons were placed in plastic containers and incubated at 25°C for 7 days. Decay lesions, identical to the original observations, developed on all inoculated lemons, while control lemons remained asymptomatic. Fungus re-isolated from the inoculated lemon was identified as P. expansum on the basis morphology and Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 sequences. Previously, Penicillium spp. including P. expansum have been reported as post-harvest pathogens on various Citrus spp. (Louw & Korsten 2015). However, P. digitatum has been reported on lemons and P. expansum has been reported on stored Kiwifruit (Actinidia arguta), Malus, and Pyrus species in China (Tai, 1979; Wang et al. 2015). To our knowledge, this is the first report of blue mold caused by P. expansum on lemons in China. References Louw, J. P., Korsten, L. 2015. Plant Dis. 99:21-30. Tai, F.L. 1979. Sylloge Fungorum Sinicorum. Sci. Press, Acad. Sin., Peking, 1527 pages. 8097 Visagie, C.M. et al. 2014. Studies. Mycol.78: 343. Wang, C. W. et al. 2015. Plant Dis. 99:1037.

The transcription factor FvHmg1 negatively regulates fruiting body development in Winter Mushroom Flammulina velutipes.

Fruiting body formation in Agaricomycetes represents the most complex and unclear process in the fungi. Mating type pathways (matA and matB) and transcription factors are important regulators in the process. Here, we report a new High-mobility-group (HMG) box domain protein FvHmg1 that acts as a negative transcription regulator in fruiting body development in Winter Mushroom Flammulina velutipes. However, the expression of Fvhmg1 in dikaryon and primordial stages was significantly lower than that of monokaryon. The Fvhmg1-RNAi mutants had a better ability of fruiting than wild type strain. Overall expression of Fvhmg1 was controlled under compatible matA and matB genes where compatible matA genes could increase its expression level, while compatible matB genes had the opposite effect. It means when two monokaryons with compatible matA and matB genes were crossed, the negatively transcription factor FvHmg1 was inhibited, and normal fully fruiting body could formation and develop. The relationship between FvHmg1 and mating type pathway would advance to understand of sexual reproduction and fruiting body development in edible mushrooms.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Cuphea hyssopifolia (Lythraceae) in Mainland China.

Cuphea hyssopifolia (Mexican heather) is a popular evergreen perennial shrub used for ornamental and medicinal purposes. Due to its high ornamental value, it is often used as a ground cover in parks and gardens in China. During February and March 2019 & 2020, powdery mildew was observed on C. hyssopifolia in the districts of Minhou and Jinshan of Fuzhou, China. Disease incidence was 70% but of low severity with only a few older leaves showing yellowing and wilting. Sparse irregular patches of white superficial powdery mildew observed on both sides of mature and young leaves. The powdery mildew fungal appressoria that occurred on epigenous hyphae, were indistinct to nipple-shaped, hyaline, and smooth. Conidiophores were erect, smooth, 80 to 210 × 10 to 12 µm, and produced two to eight crenate-shaped conidia in chains. Foot-cells of conidiophores were straight, cylindric, and 30 to 65 × 10 to12 µm. Conidia were hyaline, smooth, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 16 to 20 µm with distinct fibrosin bodies. Germ tubes were simple to forked and produced from the lateral position of the germinating conidia. No chasmothecia were observed on the surface of infected leaves. Based on the morphology of the imperfect state, the powdery mildew fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). To confirm fungal identification, total DNA was extracted (Mukhtar et al., 2018) directly from epiphytic mycelia on infected leaves collected from both districts. Internal transcribed spacer (ITS) regions and the partial large subunit (LSU) rDNA were amplified using primers ITS1/ITS4 and LSU1/LSU2 (Scholin et al. 1994, White et al. 1990), respectively. The sequences were deposited in GenBank (ITS: MW692364, MW692365; LSU: MW699924, MW699925). The ITS and LSU sequences were 99 to 100 % identical to those of P. xanthii in GenBank, (ITS: MT568609, MT472035, MT250855, and AB462800; LSU: AB936276, JX896687, AB936277, and AB936274). Koch's postulates were completed by gently pressing diseased leaves onto leaves of five healthy potted C. hyssopifolia plants that were held in a greenhouse at 24 to 30°C without humidity control. Five non-inoculated plants served as controls. Inoculated plants developed symptoms after 6 to 10 days, whereas the controls remained symptomless. The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Previously, Podosphaera sp. has been reported on C. rosea in the United Kingdom (Beales & Cook 2008) and P. xanthii on C. hyssopifolia in Taiwan (Yeh et al. 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on C. hyssopifolia in mainland China. Our field observations suggest that the P. xanthii infections would be a potential threat to the health of C. hyssopifolia in China. References: Beales, P. A., and Cook, R. T. A. 2008. Plant Pathol. 57:778. Braun, U., Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Yeh, Y. W., et al. 2021. Trop. Plant Pathol. 46:44.

Intraspecific Mitochondrial DNA Comparison of Mycopathogen Mycogone perniciosa Provides Insight Into Mitochondrial Transfer RNA Introns.

Mycogone perniciosa is the main causative agent of wet bubble disease, which causes severe damage to the production of the cultivated mushroom Agaricus bisporus around the world. Whole-genome sequencing of 12 isolates of M. perniciosa was performed using the Illumina sequencing platform, and the obtained paired-end reads were used to assemble complete mitochondrial genomes. Intraspecific comparisons of conserved protein-coding genes, transfer RNA (tRNA) and ribosomal RNA (rRNA) genes, introns, and intergenic regions were conducted. Five different mitochondrial DNA (mtDNA) haplotypes were detected among the tested isolates, ranging from 89,080 to 93,199 bp in length. All of the mtDNAs contained the same set of 14 protein-coding genes and 2 rRNA and 27 tRNA genes, which shared high sequence similarity. In contrast, the number, insertion sites, and sequences of introns varied greatly among the mtDNAs. Eighteen of 43 intergenic regions differed among the isolates, reflecting 65 single nucleotide polymorphisms, 76 indels, and the gain/loss of nine long fragments. Intraspecific comparison revealed that two introns were located within tRNA genes, which is the first detailed description of mitochondrial tRNA introns. Intronic sequence comparison within the same insertion sites revealed the formation process of two introns, which also illustrated a fast evolutionary rate of introns among M. perniciosa isolates. Based on the intron distribution pattern, a pair of universal primers and four pairs of isolate-specific primers were designed and were used to identify the five mtDNA types. In summary, the rapid gain or loss of mitochondrial introns could be an ideal marker for population genetics analysis.

First Report of Cladosporium Blossom Blight Caused by Cladosporium cladosporioides on Calliandra haematocephala in China.

Calliandra haematocephala Hassk., commonly called red powder puff, is widely cultivated as an ornamental plant in Taiwan, Hainan, Guangdong and Fujian in China (CAS, 1988). The flowers are dark crimson with conspicuous stamens, which give them the appearance of powder-puffs. Blossom blight on C. haematocephala was first observed in early January 2019 on plants grown on the university campus as well as in parks in Fuzhou city, with nearly 80% of flowers on individual plants infected. At various locations in the city, disease incidence was 100%. Symptoms appeared as grayish green fungal growth on the stamens with the entire flower eventually turning black and covered with masses of fungal spores. Fifteen single spore isolates obtained from nine necrotic stamen samples were purified and cultured on Potato dextrose agar (PDA) plates at 24 ℃.The resultant fungal colonies were olivaceous-green to olivaceous-brown and had a velvet-like appearance. Conidiophores were smooth-walled, solitary, non-nodulose, and measuring 40 to 340 × 3 to 4 µm (n=50). Ramoconidia were cylindrical-oblong or slightly curved with 0 to 3 septa, and measuring 10 to 25 × 3 to 4 µm (n=50). Conidia were smooth-walled and prolifically produced in long chains. Terminal conidia were aseptate, subglobose, ovoid to limoniform, measuring 3 to 6 × 2 to 2.5 µm (n=50). Intercalary conidia were elliptical to limoniform or subcylindrical, aseptate, measuring 5 to 12 × 2.5 to 3 µm (n=50). On the basis of its morphology, the causal organism was identified as Cladosporium cladosporioides (Bensch et al. 2010). For molecular identification, pure cultures of five single-spore isolates were used for DNA extraction. A fragment in the ITS regions of the fungal rDNA, the ACT and the TEF1-α, was amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, and EF1728 F/EF1-986R. The DNA sequences obtained from all five isolates were identical. The resulting ITS (MK720012) and ACT (MN013164), and TEFl-α (MK752020) sequences from a representative isolate MRCIM19 were 98-100% identical to the C. cladosporioides accessions (ITS: MH863979, MG228421; ACT: HM148509, JF499878, HM148532; TEFl-α: JF499872). To test pathogenicity, a spore suspension (1×105 conidia/mL) was prepared from a seven- day- old culture of isolate MRCIM19 and 10 mL of the suspension was sprayed onto six flowers on each of three C. haematocephala plants. Sterile distilled water was sprayed onto three flowers of two plants as control. The inoculated flowers were covered with plastic bags which were removed two days post inoculation. Disease symptoms were recorded on each flower at 10 days post inoculation. Based on the morpho-molecular characters, the re-isolated fungus from the inoculated flowers was C. cladosporioides. This fungus was previously reported to cause blossom blight in strawberry in the USA and Korea (Gubler et al. 1999; Nam et al. 2015). Although it has been reported from many plants (Zhang 2003) in China, this is the first report of C. cladosporioides on C. haematocephala worldwide. References Bensch, K. et al. 2010. Stud Mycol. 67:1-94. Chinese Academy of Sciences (CAS), 1988. Flora Republicae Popularis Sinicae Editorial Committee, Beijing Sci. Press., 39: 38. Gubler, W. D. et al. 1999. Plant Dis. 83:400. Nam, M. H. et al. 2015. Microbiol. 43: 354-359. Zhang Z., Ed. 2003. Flora fungorum sinicorum, Vol. 14. Cladosporium, Fusicladium, Pyricularia. Beijing Science Press. 297.

First Report of Pseudopestalotiopsis theae Causing Leaf Spot of Date Palm (Phoenix dactylifera) in China.

Date palm (Phoenix dactylifera L.) is a popular landscape tree in Fujian province, in South China. In November 2018 and June 2019, a leaf spot disease was observed on date palm in Fuzhou city. A survey of date palm plants grown in four different locations revealed that the disease incidence was almost 20%. The spots were brown with a yellow margin, 1 to 20 mm in diameter, and oval to irregular. In later stages, the spots gradually expanded and coalesced, became dry and died. For isolation, small pieces (0.5 cm2) were cut from leaf spots obtained from seven trees and disinfested with 70% alcohol. Leaf pieces were then placed onto 2% potato dextrose agar (PDA) and incubated at 25±2°C for 3 to 4 days. One fungus was consistently isolated from fifteen leaves. Fungal colonies were white with undulating margins and a light cream on the reverse side. Black globose to oblate conidiomata were irregularly distributed throughout ten-day-old colonies. The conidiogenous cells were septate, colorless, smooth-walled, straight to slightly curved, ampulliform or subcylindrical, and 6.0 to 13.5 × 1.3 to 3.0 µm [(n=50); x̄ ± SD = 9.5 ± 2× 2 ± 0.5µm]. Conidia were fusiform and five-celled with constrictions at the septa, measuring 18.5 to 31.5 × 5.0 to 7.5 µm [(n=50); x̄ ± SD = 25.5 ± 2 × 6.5± 0.2µm]. The three median cells were light to dark brown and the two end cells were colorless. Apical cells had 2 to 4 appendages ranging from 10.2 to 22.5 µm long. Basal cells had one appendage ranging from 3.5 to 5.5 µm long. The internal transcribed spacer (ITS) region of the ribosomal DNA and translation elongation factor 1-alpha (TEF1-α) gene of fungus were amplified using primers ITS1/ITS4 and EF1728F/EF1986R, respectively. Amplified products (ITS: MN294700 and TEF1-α: MN970514) showed 99% sequence identity to Pestalotiopsis sp., and Pseudoestalotiopsis theae sequences in GenBank. A comparison of MRC12 sequences with the type culture sequences (ITS: JQ683727 and TEF1-a: JQ683743) also showed high similarity, where ITS sequences exhibited only a three-nucleotide difference at the start of the sequences. No differences, however, were found between the TEF1-α sequences. On the basis of morphology and molecular characteristics, the fungus was identified as Ps. theae (Sawada) Maharachch., K.D. Hyde & Crous Steyaert (Maharachchikumbura et al. 2014). To confirm pathogenicity, five disinfested leaves on three healthy five-year-old date palm plants in a nursery (average temperature 26°C), were punctured 3 to 5 times with a sterilized needle, and then 10 to 15 mL conidial suspension (105 conidia/mL in sterilized distilled water) was sprayed over punctured areas of the leaves. For the control treatment, punctured leaves were sprayed with sterilized distilled water. All inoculated leaves plus the control were covered with plastic bags. After 10 days, brown leaf spots similar in appearance to those observed in the field appeared on all wounded leaves, and Ps. theae was successfully re-isolated; the control leaves remained asymptomatic. Previously, Ps. theae was reported on oil palm (Elaeis guineensis Jacq.) from Sierra Leone and Thailand (Turner, 1971; Suwannarach et al. 2013). To our knowledge, this is the first report of Ps. theae on date palm in China. This report expands the host range Ps. theae to date palm and underscores the potential threat of an emerging leaf spot pathogen on Phoenix species. References Maharachchikumbura, K.D., et al. 2014. Stud. Mycol. 79: 121-186. Suwannarach, N., et al. 2013. J. Gen. Plant Pathol. 79: 277-279. Turner, P.D. 1971. Phytopathol. 14: 1-58.

Characterization and expression pattern analysis of pheromone receptor-like genes in Winter Mushroom Flammulina filiformis.

Pheromone receptor-like genes (PRLGs) belong to the G protein-coupled receptors (GPCRs) family that interacts with biotic and abiotic stimulants and transmits signals to intracellular downstream pathways in eukaryotic cells. In this study, we investigated the structure and expressions patterns of PRLGs in Winter Mushroom Flammulina filiformis. Based on the alignment analysis, the structure of PRLGs was found conserved in F. filiformis strains expect few single-nucleotide polymorphism (SNP) sites. Six PRLGs were found at five different unlinked loci, scattered in the genomes of F. filiformis strains. These genes contain 2-5 introns; however, the introns were not found in the same relative positions regarding the encoded protein sequences in tested strains of F. filiformis. Three conserved motifs were identified in peptides structures of PRLGs, however, FfSte3.s6 contained only two types, suggests its difference in evolution and function. We have further analyzed the expression patterns of each PRLGs in different developmental stages of the fruiting body in F. filiformis by quantitative real-time polymerase chain reaction (qRT-PCR). The results exhibited expression variation of PRLGs at different developmental stages of the F. filiformis. Especially, FfSte3.s1 and FfSte3.s2 exhibited maximum expression level in mycelia stage. Other PRLGs exhibited high expression level in fruiting body stages. This study suggests that PRLGs could be vital genes involving in fruiting body development in F. filiformis. However, further studies could be performed to reveal their specific functional pathways in the fruiting body development.

Mitochondrial genome and diverse inheritance patterns in Pleurotus pulmonarius.

Pleurotus pulmonarius, a member of the Pleurotaceae family in Basidiomycota, is an edible, economically important mushroom in most Asian countries. In this study, the complete mitochondrial genomes (mtDNA) of three P. pulmonarius strains - two monokaryotic commercial (J1-13 and ZA3) and one wild (X1-15) - were sequenced and analyzed. In ZA3 and X1-15, the mtDNA molecule was found to be a single circle of 68,305 bp and 73,435 bp, respectively. Both strains contain 14 core protein-coding genes and two ribosomal RNA (rRNA) subunit genes. The ZA3 strain has 22 transfer RNA (tRNA) genes and nine introns: eight in cytochrome c oxidase subunit 1 (coxl), and one in the rRNA large subunit (rnl). Monokaryotic J1-13 and ZA3 mtDNAs were found to be similar in their structure. However, the wild strain X1-15 contains 25 tRNA genes and only seven introns in coxl. Open reading frames (ORFs) of ZA3/J1-13 and X1-15 encode LAGLIDADG, ribosomal protein S3, and DNA polymerase II. In addition, mtDNA inheritance in J1-13, ZA3, and X1-15 was also studied. Results showed that the mtDNA inheritance pattern was uniparental and closely related to dikaryotic hyphal location with respect to the parent. Results also show that mtDNA inheritance is influenced by both the parental nuclear genome and mitogenome in the zone of contact between two compatible parents. In summary, this analysis provides valuable information and a basis for further studies to improve our understanding of the inheritance of fungal mtDNA.

Transcriptome Profiling Revealed Multiple rquA Genes in the Species of Spirostomum (Protozoa: Ciliophora: Heterotrichea).

Adaptation to life at different oxygen tensions plays a role in protozoan ecology and controls the distribution of different species in anoxic habitats. The ciliate genus Spirostomum inhabiting fresh or low salinity water globally where these species are considered as bioindicators. Under anaerobic or low oxygen conditions, the rhodoquinol-dependent pathway has been reported in the species from the class Heterotrichea. With the help of RNA sequencing (RNAseq) data, Spirostomum spp., are suitable for deep molecular investigations on rquA for rhodoquinone (RQ) biosynthesis. In this study, Spirostomum ambiguum, Spirostomum subtilis, and Spirostomum teres collected from densely vegetated freshwater habitat in Fuzhou, China, explored the evidence of rquA. Based on transcriptome analysis, two to three RquA proteins were identified in S. ambiguum, S. teres, and S. subtilis, respectively. The presence of a key Motif-I of RquA and mitochondrial targeting signals (MTS), also confirmed the identity of these as RquA. Furthermore, Spirostomum RquA proteins could be sorted into two groups based on their conserved amino acid (CAA) residues. Phylogenetic analysis also exhibited RquA division into two subclades contained RquA1 and RquA2/RquA3 and supports two to three paralogs of rquA genes in the genomes Spirostomum spp. Additional transcriptomes and genomes analysis of Blepharisma spp., and Stentor spp., respectively, also revealed at least two paralogs of rquA in members of the class Heterotrichea. The present study provides evidence for the presence of RquA and rhodoquinol dependent fumarate reduction pathway in Spirostomum species potentially use to respire in the oxygen-depleted habitats and two to three diverse rquA genes.

Comparative Transcriptomics of Flammulina filiformis Suggests a High CO2 Concentration Inhibits Early Pileus Expansion by Decreasing Cell Division Control Pathways.

Carbon dioxide is commonly used as one of the significant environmental factors to control pileus expansion during mushroom cultivation. However, the pileus expansion mechanism related to CO2 is still unknown. In this study, the young fruiting bodies of a popular commercial mushroom Flammulina filiformis were cultivated under different CO2 concentrations. In comparison to the low CO2 concentration (0.05%), the pileus expansion rates were significantly lower under a high CO2 concentration (5%). Transcriptome data showed that the up-regulated genes enriched in high CO2 concentration treatments mainly associated with metabolism processes indicated that the cell metabolism processes were active under high CO2 conditions. However, the gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with cell division processes contained down-regulated genes at both 12 h and 36 h under a high concentration of CO2. Transcriptome and qRT-PCR analyses demonstrated that a high CO2 concentration had an adverse effect on gene expression of the ubiquitin-proteasome system and cell cycle-yeast pathway, which may decrease the cell division ability and exhibit an inhibitory effect on early pileus expansion. Our research reveals the molecular mechanism of inhibition effects on early pileus expansion by elevated CO2, which could provide a theoretical basis for a CO2 management strategy in mushroom cultivation.

Enhanced Ganoderic Acids Accumulation and Transcriptional Responses of Biosynthetic Genes in Ganoderma lucidum Fruiting Bodies by Elicitation Supplementation.

Ganoderic acids (GAs) are a type of highly oxygenated lanostane-type triterpenoids that are responsible for the pharmacological activities of Ganoderma lucidum. They have been investigated for their biological activities, including antibacterial, antiviral, antitumor, anti-HIV-1, antioxidation, and cholesterol reduction functions. Inducer supplementation is viewed as a promising technology for the production of GAs. This study found that supplementation with sodium acetate (4 mM) significantly increased the GAs content of fruiting bodies by 28.63% compared to the control. In order to explore the mechanism of ganoderic acid accumulation, the transcriptional responses of key GAs biosynthetic genes, including the acetyl coenzyme A synthase gene, and the expression levels of genes involved in calcineurin signaling and acetyl-CoA content have been analyzed. The results showed that the expression of three key GAs biosynthetic genes (hmgs, fps, and sqs) were significantly up-regulated. Analysis indicated that the acetate ion increased the expression of genes related to acetic acid assimilation and increased GAs biosynthesis, thereby resulting in the accumulation of GAs. Further investigation of the expression levels of genes involved in calcineurin signaling revealed that Na+ supplementation and the consequent exchange of Na+/Ca2+ induced GAs biosynthesis. Overall, this study indicates a feasible new approach of utilizing sodium acetate elicitation for the enhanced production of valuable GAs content in G. lucidum, and also provided the primary mechanism of GAs accumulation.