A study on the genus Candolleomyces (Agaricales: Psathyrellaceae) from Punjab, Pakistan.

Many basidiomata of the genus Candolleomyces were found on sandy and loamy soil from the Indus Riverbed, Kot Addu District. A phylogenetic study was conducted to examine the occurrence of Candolleomyces sindhudeltae sp. nov. using a combination of ITS and LSU regions. Our morphological, anatomical, and phylogenetic studies indicated the novelty of Candolleomyces sindhudeltae sp. nov. The distinguishing features of C. sindhudeltae are convex to campanulate and areolate pileus with scalloped to cracked cap margins, branched, and pale reddish lamellae, greenish-brown ellipsoid to ovoid basidiospores, polymorphic cheilo, and caulocystidia. The novel taxa formed independent phylogenetic relationships within the genus Candolleomyces. The addition of our new species to the genus Candolleomyces makes us confident that the genus was separated from Psathyrella accurately.

Estimation of some trace metals, bioactive compounds, curative antimicrobial and antioxidant agents from Russula foetens and Russula cf. foetentoides.

The current study is an attempt to estimate minerals, antioxidant and antimicrobial activities of two medicinal mushrooms (Russula foetens and Russula cf. foetentoides) along with their mycochemicals. Among the metal analysis, iron was most abundantly (7.428mg/g) present in R. cf. foetentoides. The mycochemical screening revealed the presence of active secondary metabolites and phenolic composition by HPLC showed five stable compounds with maximum concentration of gallic acid (22.31%). The highest antibacterial activity (27.8±0.152mm) was displayed in ethanol macerate of R. foetens in contradiction of Staphylococcus aureus. Similarly, highest antifungal potential was exhibited by R. foetens (ethanolic macerate) against Fusarium equiseti with zone of inhibition 23±0.572mm. The antioxidant potential was assessed by five assays and the R. foetens exhibited maximum % inhibition (43.045±0.037%) by DPPH radical protocol. Maximum % inhibition by ABTS protocol was exhibited by R. cf. foetentoides i.e., 88.057±0.021. The highest reducing potential was revealed in R. foetens (0.684±0.001mmol/L of FeSO4). The highest flavonoids and total phenolic contents (TPC) were shown by R. cf. foetentoides i.e., 0.115±0.002mg/100g of Catechin and 0.064±0.001mg/100g of GAE (gallic acid equivalents), respectively. All the obtained results were analyzed statistically by applying the ANOVA to determine the significant and non-significant ranges.

Short Communication- Estimation of trace elements and in vitro biological activities of lichens extracts.

Infectious diseases caused by etiological agents are still a major threat to public health. Their impact is particularly large in developing countries due to relative unavailability of medicine and the emergence of widespread drug resistance. In the current research, trace metals were detected in lichens species through inductively coupled plasma atomic emission spectroscopy. The antimicrobial potency of Pseudevernia furfruracea, Physcia species, Dermatocarpon vellerum and Parmellia species (lichens) extracts have been investigated against three local clinical bacterial isolates i.e. Escherichia coli, Pseudomonas aeruginosa and Staphylococcus epidermidis through various agar disc and well diffusion methods. The antioxidant potential effect was also evaluated by DPPH and ABTS.+ free radical scavenging methods. Phytochemical constituents were screened through thin layer chromatography (TLC) and qualitative methods. Methanolic extract of P. furfruracea, Physcia spp, and D. vellerum showed a significant inhibition of S. epidermidis (14.3±1.7mm, 12.3±2.0mm, and 11.3±0.9mm) by pouring method of disc diffusion. Moderate zone of inhibition (8.0±1.4 mm) against S. epidermidis was observed by methanolic extract of Parmellia spp, through spreading method. All the results were evaluated by ANOVA and LSD tests at p<0.05. The diethyl ether extracts showed considerable antioxidant potential activity with 80%, 81%, 79% and 66%. Thin layer chromatography profiling gave us the idea about the presence of phytochemical constituents such as tannins, phenols, saponins, and terpenoids. Various Rf values on silica gel plates provided the valuable clues about polarity and the selection of solvents for separation of phytochemicals. Significant inhibition of E. coli was also observed through TLC-Bioautography. The findings revealed the considerable inhibitory and antioxidant effect of lichens may be due to the presence of bioactive compounds. Therefore, lichens could be a potential source of new antimicrobial and antioxidant agents.

Multigene phylogeny and morphological descriptions of five species of Agaricus sect. Minores from subtropical climate zones of Pakistan.

The genus Agaricus includes more than 500 species mostly containing the edible and cultivated species worldwide. As part of the ongoing studies on the biodiversity of genus Agaricus in Pakistan, our objective was to focus on A. sect. Minores which is the largest section of the genus. In the first phylogenetic analyses based on the ITS region of the nuclear ribosomal DNA, our sample included specimens of 97 named species, 27 unnamed species, and 31 specimens (29 newly generated sequences in this study) from subtropical climate zones of Pakistan that likely belong to this section based on their morphology. The 31 specimens grouped into five distinct, well-supported clades corresponding to five species: A. glabriusculus already known from Pakistan and India, A. robustulus first recorded from Pakistan and briefly described here but already known from Bénin, Malaysia, China, and Thailand, and three possibly endemic new species described in detail A. badiosquamulosus sp. nov., A. dunensis sp. nov., and A. violaceopunctatus sp. nov. The sixth species currently known in Pakistan, including A. latiumbonatus also found in Thailand, were included in a multigene tree based on ITS, LSU, and Tef-1α sequence data. They all belong to a large pantropical paraphyletic group while most temperate species belong to a distinct clade, which includes about half of the species of the section. The current study aims to propose three novel species of genus Agaricus based on comprehensive morphological as well as molecular phylogenetic evidences from Pakistan.

Four new species of Candolleomyces (Psathyrellaceae) from the Punjab Plains, Pakistan.

The purpose of this study is to explore the genus Candolleomyces from Pakistan. Fungal surveys were conducted in different sites of the Punjab plains during the 2016-2022 period. Several specimens belonging to the family Psathyrellaceae were collected. Traditional morphology and multigene phylogenetic analyses confirmed the novelty and placement of the species in genus Candolleomyces. The phylogenetic study of the nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) and partial 28S rRNA regions confirmed the distinct positions of four new taxa, Candolleomyces iqbalii, C. kotadduensis, C. pakistanicus, and C. parvipileus, in the genus. Currently, the genus consists of 43 species worldwide. However, with the inclusion of these four species, the total number has risen to 47. Detailed descriptions, phylogeny, comparisons, and illustrations are provided.

New species and new records of genus Buellia (lichenized Ascomycota, Caliciaceae) from Pakistan using electron microscopy and DNA barcoding techniques.

Three new species Buellia darelensis, B. densitheca, B. kashmirensis and two new records B. elegans and B. taishanensis are added to the lichen biota of Pakistan. Buellia darelensis and B. kashmirensis share the same habitats found on the rock while B. densitheca colonized on bark of Pinus hardwoods in the Himalayan forest, Pakistan. Morphological, chemical, and phylogenetic analyses were carried out to elucidate the placement of these species and to support the delimitation of the new taxa. Detailed descriptions and figures for the species are given, and a key to all known buellioid species from Pakistan is provided. RESEARCH HIGHLIGHTS: During recent explorations of lichens from different regions of Pakistan, we observed specimens that could not be readily assigned to any known species. A phylogenetic analysis of the internal transcribed spacer nrDNA region confirms their position within the genus Buellia, and morphological data showed distinctiveness of three species from other known species of the genus. We therefore describe these specimens as new species to science, and two species are as new records for the country. Pakistan exhibits a large altitudinal variation, with climatic conditions and a diverse vegetation that supports a diverse and conspicuous lichen biota. The nature reserves have abundant biological resources, and it is expected that more new species of lichen may be discovered in the future.

Culturability, Cultivation Potential, and Element Analysis of the Culinary-Medicinal Cauliflower Mushroom Sparassis latifolia (Agaricomycetes) from Pakistan.

Sparassis Fr. is a recognized nutritionally and therapeutically significant genus of mushrooms. Its species has the potential to meet the nutritious and healthy food needs of the growing population but unfortunately due to their tough, laborious and undefined cultivation conditions, their domestication is very rare and not properly described before. In this study, first time the detailed culturabilty and cultivation potential of a wild S. latifolia species from Pakistan was evaluated. Among the different media used, maximum cultural growth was observed on pine needles extract agar (PEA) medium at 24°C. Cultured strains on PEA medium were used to spawn on wheat, sorghum and barley grains. Sorghum grains at 24°C were found as the best combination for spawn production of this mushroom. Total yield efficacy was investigated on a variety of substrates. A mixed substrate of Morus sawdust and pine sawdust at 20°C showed the optimum yield. Element analysis of wild and cultivated strain was carried out and revealed that this mushroom is enriched with macronutrients. These results showed that S. latifolia has great artificial growth potential. Its domestication can compete with commonly growing mushrooms in nutritional and pharmaceutical attributes.

Antioxidant Potential and Stability of Oxidative Sunflower Oil by Using the Ethanolic Extracts of Saprophytic Edible Cultivable Mushrooms from Pakistan.

During this study, ethanol extracts of four edible mushrooms i.e., Macrocybe gigantea, Podaxis pistillaris, Lentinus tigrinus, and Pleurotus cystidiosus were prepared and investigated by TFC, TPC, FRAP, ABTS and DPPH for their antioxidant potential. The extracts were also used to check the oxidative stabilization of sunflower oil for a period of 45 d using standard methods i.e., peroxide value, free fatty acid contents and iodine value. M. gigantea extract showed the maximum flavonoids and phenolic contents and exhibited the maximum antioxidant potential among the four edible mushrooms. BHA was observed to have maximum oil stabilization potential followed by the ethanol extract of M. gigantea of 1000 ppm concentration, BHT and extract of 1000 ppm concentration of P. pistillaris. Thus, it is concluded from this study that ethanolic extracts of these mushrooms can be used as a healthy supplement to stabilize oil by replacing synthetic antioxidants that are harmful to humans.

Domestication of a magic therapeutical wine glass fungus (Podoscypha petalodes) from Pakistan.

The Podoscypha is a recognized therapeutically significant genus of mushrooms. A Podoscypha species under a Morus sp. Was found from the largest man-made forest Changa-Manga, Kasur during the exploration of fungal flora of Pakistan. Detailed morpho-anatomical and phylogenetic analysis identified it as P. petalodes, a part of common native flora of many regions of the world like Pakistan. The culturabilty and cultivation potential of this fungus was assessed for the first time using different media and substrates. Maximum cultural growth was observed on the Compost Extract Agar (CEA) medium at 28 °C. Mycelium of cultured strains on CEA medium was used for the spawn production on wheat, sorghum and barley grains. Cultivation potential in the form of spawn running period, harvesting time duration and yield was investigated on variety of substrates. A mixed substrate of sawdust and tea waste at 28 °C showed the optimum yield. Tea-waste was used as the casing material in all substrates and proved very effective. These results depicted that Podoscypha petalodes possesses the cultivation potential. Its cultivation on large scale can solve the major health concerns of the growing population. It would provide the people easy accessibility of economical, effective and natural medicine throughout the year that restrict in case of only natural production at specific time of the year.

Appraisal of Antioxidant Potential and Natural Bioactive Compound Determination through HPLC from Two Saprotrophic Amanita (Agaricomycetes) Species from Pakistan.

The present study was carried out to investigate and identify bioactive compounds along with antioxidant capacity, total flavonoids and total phenolic contents from two saprophytic Amanita species, i.e., mushrooms A. manicata (Berk. & Broome) Pegler and A. nana Singer. Antioxidant potential was assessed by DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging and ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assay, total phenolics, and flavonoids. Both mushrooms were found to possess antioxidants and wide range of phenolics and bioactive compounds. There was maximum percent inhibition (83.2 ± 0.120%) on DPPH by A. manicata. However, maximum percent inhibition using ABTS was found to be 79.5 ± 0.251% by A. nana. Similarly, A. nana possesses maximum amount of both flavonoids and phenolics i.e., 0.3473 ± 0.0088 mg/100 g of catechin and 0.097 ± 0.0011 mg/100 g of GAE equivalent, respectively. Both mushrooms exhibited a variety of natural compounds such as P-coumaric acid, M-coumaric acid, benzoic acid, ferulic acid etc. Ferulic acid, chlorogenic acid, and cinnamic acid were also detected in A. manicata. A. manicata exhibited best and greater antiradical potential than A. nana due the presence of excessive natural bioactive compounds. From the reported results, it was revealed that both inedible mushrooms could be a potential source of antioxidants and secondary metabolites and might be used for making novel drugs in future by pharmaceutical industries.

Systematic characterisation, and effect of nutritional and physical parameters on culturability, laccase production and dye decolorisation potential by P. pistillaris from Pakistan.

Podaxis pistillaris is neutraceutically, cosmoceutically and medicinally recognised macrofungus. During this research work, this edible mushroom was systematically characterised. Its culturability, laccase production, and dye decolorisation potential were evaluated and optimised. Among the different media tested, PDA proved as most efficient medium for culturability of P. pistillaris. Conditions for laccase production were optimised in submerged state fermentation. Maximum laccase secretion was noted after 14th day of Incubation at 35 °C with 130 rpm and 5 pH of medium. Fructose and ammonium-phosphate was found as best carbon and nitrogen source, while wheat straw revealed as good ligno-cellulosic source for strengthening laccase production. Congo-red dye decolorisation capability by crude laccase enzyme was evaluated and found maximum decolorisation potential (92.2%) after 288h of incubation. From this pilot study, it was confirmed that this edible macrofungus has culturability, laccase production and dye decolorisation attributes that will further contribute in delignification, biosorption and bioremediation.

Extrapolating Qualitative Mycochemical Profiling and Therapeutic Potential of Two Mycorrhizal Amanita Species (Agaricomycetes) from Pakistan.

The present study was an attempt to evaluate the antimicrobial and anthelmintic potential of two Amanita species, i.e., A. orsonii and A. glarea, along with their qualitative mycochemical screening. Maceration technique was adopted to make crude extracts in non-polar (petroleum ether and chloroform) and polar (ethanol and distilled water) solvents. Qualitative mycochemical screening revealed the presence of a variety of secondary metabolites like terpenoids, flavonoids, tannins, alkaloids, saponins, and cardiac glycosides. Antimicrobial activities were carried out by using agar well diffusing method against four bacterial and one fungal strain. The antibacterial potential ranged between 4.86 ± 0.088 mm and 34.83 ± 0.166 mm with maximum inhibition zone exhibited by petroleum ether extract against Pseudomonas fluorescens and least potential by distilled water macerate of A. orsonii against the Escherichia coli. The antifungal activity ranged between 14.5 ± 0.288 and 24.76 ± 0.145 mm, with the highest potential provided by chloroform extract of A. orsonii and least capability put forward by A. glarea by petroleum ether extract against Fusarium solanii. Antibiotics and antimycotic discs were used as standard and some of the crude extracts showed bigger zone of inhibition as compared to standard. Different concentrations of ethanolic extracts of both mushrooms were subjected to anthelmintic potential against parasite Haemonchus contortus. All the crude extracts were more potent than standard oxfandazole used. Anthelmintic potential ranged between 9.9 ± 0.057 and 54.93 ± 0.033 minutes for death of parasite, while the paralyzed time ranged between 4.86 ± 0.088 and 24.86 ± 0.088 minutes. From the results obtained it was concluded that both mushrooms can be used as potential source of curative antibacterial, antifungal, and anthelmintic agents against several diseases that might be used in pharmaceutical industries for making medicines and to screen out secondary metabolites in future.

Dose optimization of silicon for boosting arbuscular mycorrhizal fungi colonization and cadmium stress mitigation in maize (Zea mays L.).

The foliar applied silicon (Si) has the potential to ameliorate heavy metals, especially cadmium (Cd) toxicity; however, Si dose optimization is strategically important for boosting the growth of soil microbes and Cd stress mitigation. Thus, the current research was performed to assess the Si-induced physiochemical and antioxidant trait alterations along with Vesicular Arbuscular Mycorrhiza (VAM) status in maize roots under Cd stress. The trial included foliar Si application at the rate of 0, 5, 10, 15, and 20 ppm while Cd stress (at the rate of 20 ppm) was induced after full germination of maize seed. The response variables included various physiochemical traits such as leaf pigments, protein, and sugar contents along with VAM alterations under induced Cd stress. The results revealed that exogenous application of Si in higher doses remained effective in improving the leaf pigments, proline, soluble sugar, total proteins, and all free amino acids. Additionally, the same treatment remained unmatched in terms of antioxidant activity compared to lower doses of foliar-applied Si. Moreover, VAM was recorded to be at peak under 20 ppm Si treatment. Thus, these encouraging findings may serve as a baseline to develop Si foliar application as a biologically viable mitigation strategy for maize grown in Cd toxicity soils. Overall, the exogenous application of Si helpful for reducing the uptake of Cd in maize and also improving the mycorrhizal association as well as the philological mechanism and antioxidant activities in plant under cadmium stress conditions. Also, future studies must test more doses concerning to varying Cd stress levels along with determining the most responsive crop stage for Si foliar application.

Mycelial Growth, Basidiocarp Production and Mineral Analysis of the Edible Lentinus squarrosulus (Agaricomycetes) from the Subtropical Part of Pakistan.

Lentinus squarrosulus (Mont.) Singer, a wild edible macrofungus, possesses confirmed organoleptic, nutritional, and medicinal attributes. During the exploration of fungal biota of Punjab, Pakistan, L. squarrosulus was collected under the Morus alba from University of the Punjab, Lahore. For the domestication of this wild significant species, its culturability and cultivation potential was assessed by using different synthetic culture media and substrates. Among these different media used, maximum cultural growth was observed on the malt-extract agar (MEA) medium at 35°C followed by the potato-dextrose agar (PDA), compost extract agar (CEA), glucose-peptone agar (GPA), and Sabouraud-dextrose agar (SDA). Cultured strains on MEA medium were used to produce spawning material on wheat, sorghum, and barley grains. Sorghum grains at 35°C were found to be the best combination for spawn production of this fungus. Yield and biological efficiency were investigated on a variety of substrates. A mixed substrate of wheat straw and tea waste at 30°C showed the optimum yield. Tea-waste medium was used as the casing material and proved very effective. Mineral analysis of the wild and cultivated strain was carried out and revealed that both forms were enriched with macronutrients. These results showed that the L. squarrosulus strain from Pakistan possesses good nutrient and growth potential and its domestication can compete with nutritional and medicinal properties of commonly growing mushrooms.

Tulostoma loonbanglaense: A new species from Azad Jammu and Kashmir using light and scanning electron microscopy and DNA barcoding technique.

Tulostoma loonbanglaense sp. nov. is described from Azad Jammu and Kashmir, Pakistan. ITS sequences confirm its position within the genus Tulostoma, and suggest that it is separate from other identified species of this genus. The species has globose to subglobose, with wide, solitary and appressed spines basidiospores, the basidiospores are relatively small, 4.39 × 4.23 µm, that make it distinguished from the related species. Sequences of nr ITS region of the newly described species nested as a distinct taxon in both phylogenetic analyses of the current study.

Proximate Analysis and In Vitro Biological Activities of Cauliflower Mushroom, Sparassis crispa (Agaricomycetes), from Pakistan.

This study evaluates the proximate analysis and antibacterial and antioxidant potential of the edible mushroom, Sparassis crispa. The preliminary mycochemical analysis revealed the presence of secondary metabolites such as saponins, terpenoids, flavonoids, tannins, and cardiac glycosides. Proximate analysis was performed to estimate the presence of some essential and nonessential metals in the sample. Among the minerals analyzed, zinc was in the maximum range (1.156 mg/g) compared to the other elements. The antioxidant potential of S. crispa ethanolic extract was assessed by using five assays: 1) 2,2'-azino-bis-3-ethylbenzothiazoline-6-suphonic acid, 2) ferric reducing antioxidant power, 3) 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 4) total flavonoid content, and 5) total phenolic content. The DPPH radical scavenging assay confirmed the highest percent inhibition of the extract (56.43% ± 0.21%). Antibacterial activity of the mushroom sample was tested against the selected Gram-negative and Gram-positive bacterial strains by using the agar well diffusion method. S. crispa ethanolic extract exhibited maximum antibacterial activity with an inhibition zone of 19.66 ± 0.88 mm against Escherichia coli compared to other bacterial strains. From these results, it could be assessed that S. crispa is a promising source of new antibacterial and antioxidant agents.

Evaluation of Mycochemical Analysis and In Vitro Biological Activities of Some Russula Species (Agaricomycetes) from Pakistan.

In this study, mycochemical screening was performed to estimate the in vitro biological activities and antimicrobial and antioxidant potential of Russula aeruginea and R. brevipes. Crude extracts of these mushroom species were obtained by using maceration techniques. Qualitative mycochemical analysis showed the presence of a variety of secondary metabolites such as terpenoids, alkaloids, flavonoids, reducing sugars, and anthraquinones. Antimicrobial activity of crude extracts was assessed by using agar well diffusion methods against virulent bacterial and fungal strains. Ethanolic extract of R. brevipes displayed maximum antibacterial activity, with a 28.4 ± 0.953 mm zone of inhibition against Bacillus subtilis. Likewise, ethanolic extract of R. aeruginea exhibited enhanced antifungal activity, with a 24.6 ± 0.333 mm zone of inhibition against Fusarium equiseti. The antioxidant potential of the ethanolic extracts was assessed using five assays: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power assay (FRAP), total flavonoid content (TFC), and total phenolic content (TPC). The maximum percent inhibition for R. aeruginea was 87.069 ± 0.007 mg/mL on ABTS radicals. In contrast, the maximum percent inhibition for R. brevipes was 42.166% ± 0.023% and 0.571 ± 0.005 mmol/L of FeSO4 using the DPPH and FRAP methods, respectively. TFC was the same for both mushrooms (0.113 ± 0.002 mg/100 g of catechin), and the maximum TPC was 0.066 ± 0.00 mg/100 g of gallic acid equivalents. From these results, it was concluded that R. aeruginea and R. brevipes could be a source of new antimicrobial and antioxidant agents and may be useful for the development of novel drugs in the future.

Mycochemical Screening and Study of Medicinal Properties of Two Amanita Species (Agaricomycetes) from Pakistan.

Fresh basidiocarps of Amanita cinnamomescens and A. pakistanica were collected from Ayubia-Khanspur, Pakistan, during the 2018 monsoon season. Basidiocarps of A. cinnamomescens and A. pakistanica were evaluated for their mycochemicals, mineral composition, and antioxidant and antimicrobial activities. The percentage yield of extracts ranged from 4.13% to 18.20%. The extracts contained noticeable total phenolic contents (0.043 ± 0.02 to 0.046 ± 0.01 mg/g) and total flavonoid contents (0.090 ± 0.004 to 0.0935 ± 0.003 mg/g) and good radical scavenging ability according to the ABTS assay (79.74% ± 0.03% to 85.34% ± 0.02%) and the DPPH radical assay (35.77% ± 0.01% to 44.77% ± 0.003%). In addition, the tested extracts showed substantial antimicrobial activity, which ranged from 10 ± 0.33 to 32.66 ± 0.33 mm. Both mushrooms were also analyzed for their mineral content (sodium, potassium, calcium, nickel, cobalt, copper, zinc, manganese, chromium, and iron). It was concluded that A. cinnamomescens and A. pakistanica can be used as a potential source for formulation of dietary functional foods and pharmaceuticals with antioxidant and antimicrobial effects. To our knowledge, this is the first report on in vitro biological activities and mycochemical analysis of A. pakistanica and A. cinnamomescens from Pakistan.

An overview of Agaricus section Hondenses and Agaricus section Xanthodermatei with description of eight new species from Pakistan.

In a recent revision of the genus Agaricus, A. section Xanthodermatei was split into two sections A. sect. Hondenses and A. sect. Xanthodermatei. Our objectives were to investigate the species diversity of both sections in Pakistan and to give an overview of the major clades. Phylogenetic analyses based on the combined nucLSU, ITS and TEF1 dataset from 35 specimens of both sections revealed three major clades. Analyses based on ITS dataset and 106 specimens, including 33 from Pakistan, reveal eight new species and one new record species. These nine species are described in detail. It is noteworthy that intraspecific variability as well as interspecific variability between closely related species were very low in ITS sequences in many cases. In the case of the two new species A. xanthochromaticus and A. griseovariegatus, TEF1 sequence data were much more efficient than ITS to distinguish these species from each other. The other new species are A. atroumbonatus, A. fumidicolor, A. macropeplus, A. parviniveus, A. swaticus and A. bambusetorum. The latter is the only new species of A. sect. Hondenses in which it is morphologically atypical and also the unique (sub)tropical species. Agaricus gregariomyces is recorded for the first time in Pakistan. In addition, brief descriptions are provided not only for A. bisporiticus, A. endoxanthus and A. punjabensis, which are reported again in Pakistan, but also for A. californicus, which is reported for the first time in Spain and outside North America. In total 12 species of both sections were reported in Pakistan and half of them were from subtropical climatic areas, underlining the contribution of the climatic diversity to the high species richness in this country.