Advancements in genetic studies of mushrooms: a comprehensive review.

Genetic studies in mushrooms, driven by innovations such as CRISPR-Cas9 genome editing and RNA interference, transform our understanding of these enigmatic fungi and their multifaceted roles in agriculture, medicine, and conservation. This comprehensive review explores the rationale and significance of genetic research in mushrooms, delving into the ethical, regulatory, and ecological dimensions of this field. CRISPR-Cas9 emerges as a game-changing technology, enabling precise genome editing, targeted gene knockouts, and pathway manipulation. RNA interference complements these efforts by downregulating genes for improved crop yield and enhanced pest and disease resistance. Genetic studies also contribute to the conservation of rare species and developing more robust mushroom strains, fostering sustainable cultivation practices. Moreover, they unlock the potential for discovering novel medicinal compounds, offering new horizons in pharmaceuticals and nutraceuticals. As emerging technologies and ethical considerations shape the future of mushroom research, these studies promise to revolutionize our relationship with these fungi, paving the way for a more sustainable and innovative world.

Ceratocystis fimbriata Causing Postharvest Fruit Rot of Pear in Punjab, Pakistan.

Pear (Pyrus communis) is an important deciduous fruit cultivated on a worldwide scale including Pakistan. During August 2021, a postharvest fruit rot disease was observed on several pears at various farmers market in Okara- a district of Punjab Province, Pakistan. The incidence of the disease varied from 7 to 20% with 35% disease severity. Necrotic spots (10 to 20 mm diameter) were first observed on the infected pear fruit. The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium. The isolates were obtained from the symptomatic tissues (n = 18) incubated on carrot discs that had been surface sterilized in 100-ppm streptomycin solution. After consistent sporulation of a fungus on the carrot discs, the ascospore masses formed at the tip of perithecia were transferred to malt extract agar (MEA). Primary conidia were cylindrical and hyaline (7 to 11 × 4 to 7 µm) and secondary conidia were hyaline and barrel-shaped (7 to 12 × 5 to 8 µm). Endoconidiophores with primary conidia were (12 to 27 × 2.6 to 5.5 µm). Perithecia produced on carrot discs were dark brown to black, and the base was 157 to 278 µm in diameter. Ascomatal necks were 512 to 656 µm long, dark brown to black, lighter in color at apices, tapering from base (23 to 45 µm diameter) to apex (13 to 24 µm diameter). Ostiolar hyphae were 41 to 79 µm long. Ascospores were hyaline, hat shaped, 3 to 4 µm long, and accumulated in a sticky matrix at the tips of perithecial necks. Mycelium was initially hyaline but became dark greenish brown after 7 days. Dark brown, thick-walled aleuroconidia (13 to 19.5 × 9 to 14 µm) appeared on culture plates after 2 months. Based on morphological characteristics, the fungus was identified as Ceratocystis fimbriata (Engelbrecht, 2005; Suwandi et al. 2021). To further confirm species identification, genomic DNA of two representative isolates (UO-05 and UO-06) was obtained using an extraction kit. The internal transcribed spacer (ITS) region was amplified using ITS1/4 (White et al. 1990). A BLAST search with GenBank accession nos. OR185451 and OR185456 indicated 99 to 100% identity with several C. fimbriata including type species (MH856050.1; KC493160.1; MT560374.1). Pathogenicity tests were conducted by inoculating nine disease-free pear (cv. Concord) fruit after disinfesting in 75% ethanol. A prepared spore suspension (1.0 × 106 spores/ml) was dropped on the wounds (a depth of 1 mm diameter) on the pear surface, which were made by a sterilized needle. 10 µl of a prepared spore suspension was dropped onto nine pears. Sterile water (10 µl) was dropped on the wounded sites of nine pear fruits as negative controls and all fruits were incubated in a growth chamber at 30/26°C (day/night, 90% relative humidity). Symptoms similar to those on the naturally infected fruits began after 4 to 5 days of inoculation, while controls remained healthy. The fungal isolates recovered from inoculated pears were morphologically identical to the C. fimbriata isolates originally recovered from symptomatic fruits fulfilling Koch's postulates. The pathogen has been reported to cause postharvest fruit rot of passion fruit and cucumber (Firmino et al. 2016; Li et al. 2019). To our knowledge, this is the first report of C. fimbriata causing fruit rot of pear in Punjab Province. The detection of this disease will help pear growers to take actions to monitor and prevent disease outbreak as well as develop an effective management practice when it occurs.

First report of wheat dwarf bunt caused by Tilletia controversa Kühn in Pakistan.

Wheat (Triticum aestivum L.) is critical to food security worldwide. Wheat dwarf bunt is caused by Tilletia controversa Kühn and can cause 70-80% losses under severe condition (Trione et al. 1989; Xu et al., 2021). In May 2022, we observed dwarf bunt disease in six fields grown with spring cultivar (Glaxy-13) in District Swat, KPK-Pakistan. Infected plants had mottling and flecking on leaves, a greater number of tillers and were smaller than healthy plants. Diseased wheat head spikes were larger, wider and thicker, had bunted kernels (sori) filled with brown-black teliospores and a strong odor like that of rotten fish. Individual fields showed 10% infected plants while no dwarf bunt was recorded in nearby fields. About 150 heads exhibiting bunted kernels were collected among the six fields. Kernels were surface sterilized with 30% NaClO for 5 min after crushing by a centrifuge machine and washed with ddH20 three times. The teliospore suspension (1×106 spores/mL) was spread on 2% soil agar plates in a growth chamber (MLR 352 H, Panasonic, USA) and incubated at 5°C with 60% relative humidity for 60 days to test for T. controversa germination or at 16°C and 60% relative humidity for 15 days (MLR 352 H, Panasonic, USA) to test for T. caries and T. laevis germination. Teliospores germinated only on plates kept at 5°C. Teliospores were morphologically identified as a T. controversa from the infected samples. They ranged in size from 15.0 to 20.5 µm diam. and the walls had deep reticulations surrounded by a transparent sheath, differing from T. laevis which has smooth teliospores and T. caries which has no sheath and reticulations on the surface (Mathre 1996). To further confirm Tilletia spp. identification, genomic DNA of our two isolates (gmd123 and gmd1234) was obtained using an extraction kit (TransGen, Beijing, China). The internal transcribed spacer (ITS) region was amplified by using ITS1/4 (White et al. 1990). A BLAST search with GenBank accession no. OR366448 and OR366450 provided additional evidence the isolates belong to the complex of species that includes the three bunt species causing diseases on wheat, with 100% matches to verified sequences for T. controversa (eg. EU257561) but also to T. laevis and T. caries. Based on disease symptoms, teliospore morphology, germination at 5°C but not at 16°C, the bunt fungus was identified as T. controversa. To fulfill Koch's postulates, 10 mL (106 spores/mL) of germinated teliospores were injected into rhizosphere soil of Galaxy-13 cultivar at 2 leaves unfolded growth stage (Zadoks 12) and 2 mL (106 spores/mL) were injected into heads of same plants at growth stages Zadoks 61-65 with a syringe. Plants injected with sterile ddH2O were used as a control. Inoculated plants were grown in a growth chamber at 8°C with 50% humidity and 24 h light. After one month at the ripening stage, the bunted kernels of the inoculated plants were filled with black teliospores releasing a fishy smell, and the control plants did not have bunted kernels. Under an optical microscope, teliospores from the inoculated plants had reticulation surface and were measured 15 to 20.5 µm in diameter, similar to the teliospores of bunt heads from the fields. To the best of our knowledge, this is the first report of T. controversa causing dwarf bunt in district Swat, KPK-Pakistan. Because the pathogen is seedborne and soilborne, the disease may become a high risk to wheat production in Pakistan. Therefore, detection of this pathogen is very important to control the disease on time.

Taxonomy, biological characterization and fungicide sensitivity assays of Hypomyces cornea sp. nov. causing cobweb disease on Auricularia cornea.

Auricularia cornea is an important edible mushroom crop in China but the occurrence of cobweb disease has cause significance economic loss in its production. The rate of disease occurrence is 16.65% all over the country. In the present study, a new pathogen Hypomyces cornea sp. nov. was found to cause the cobweb disease. In July 2021, three strains of fungal pathogen were isolated from infected fruiting bodies and identified as H. cornea based on morphological studies and molecular phylogenetic analysis of internal transcribed spacer (ITS) of nuclear ribosomal DNA, mitochondrial large subunit (LSU) of rRNA and the partial translation elongation factor 1-alpha genes. The representative isolates of the pathogenic Hypomyces species used to perform pathogenicity test with spore suspension that caused similar symptoms as those observed in the cultivated field, and same pathogens could be re-isolated, which fulfill Koch's postulates. The typical biological characterization was examined of the serious pathogen to determine its favorable growth conditions, including suitable temperature, pH, carbon, nitrogen sources and light conditions. The findings revealed an optimum temperature of 25 °C, pH of 6, and soluble starch and peptone as the preferred carbon and nitrogen sources, respectively. The hyphal growth inhibition method was used for primary in vitro screening test of seven common fungicides, and the most suitable fungicide is Prochloraz manganese chloride complex, the EC50 values of cobweb pathogen and mushrooms were 0.085 µg/mL and 2.452 µg/mL, respectively. The results of our research provide an evidence-based basis for the effective prevention and treatment of A. cornea cobweb disease.

Pezizomycotina species associated with rotten plant materials in Guizhou Province, China.

Nine Pezizomycotina strains were isolated from rotten dead branches and leaves collected from Guizhou Province. To obtain their accurate taxonomic placement, we provided the morphological characteristics of conidiophore cells and conidia. Phylogenetic relationships, based on ITS, rpb2, SSU, LSU and tub2 gene sequences, confirmed our strains represented three novel species, Peglioniafalcata, Neoascochytapseudofusiformis and Neomicrosphaeropsiscylindrica. Peglioniafalcata produced falcate conidia and Neoa.pseudofusiformis generated fusiform conidia, while Neom.cylindrica possessed cylindrical conidia. The phylogenetic results also supported them as novel taxa. All the new species in the present study were found as saprophytic on forest litter with high rainfall, which suggest they may have a certain effect on nutrient decomposition and redistribution in forest ecosystems. Thus, it opened a way for further research on related ecological roles and their application production.

Evolution and related pathogenic genes of Pseudodiploöspora longispora on Morchella based on genomic characterization and comparative genomic analysis.

True morels (Morchella) are globally renowned medicinal and edible mushrooms. White mold disease caused by fungi is the main disease of Morchella, which has the characteristics of wide incidence and strong destructiveness. The disparities observed in the isolation rates of different pathogens indicate their varying degrees of host adaptability and competitive survival abilities. In order to elucidate its potential mechanism, this study, the pathogen of white mold disease from Dafang county, Guizhou Province was isolated and purified, identified as Pseudodiploöspora longispora by morphological, molecular biological and pathogenicity tests. Furthermore, high-quality genome of P. longisporus (40.846 Mb) was assembled N50 of 3.09 Mb, predicts 7381 protein-coding genes. Phylogenetic analysis of single-copy homologous genes showed that P. longispora and Zelopaecilomyces penicillatus have the closest evolutionary relationship, diverging into two branches approximately 50 (44.3-61.4) MYA. Additionally, compared with the other two pathogens causing Morchella disease, Z. penicillatus and Cladobotryum protrusum, it was found that they had similar proportions of carbohydrate enzyme types and encoded abundant cell wall degrading enzymes, such as chitinase and glucanase, indicating their important role in disease development. Moreover, the secondary metabolite gene clusters of P. longispora and Z. penicillatus show a high degree of similarity to leucinostatin A and leucinostatin B (peptaibols). Furthermore, a gene cluster with synthetic toxic substance Ochratoxin A was also identified in P. longispora and C. protrusum, indicating that they may pose a potential threat to food safety. This study provides valuable insights into the genome of P. longispora, contributing to pathogenicity research.