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.

Exploring Trichoderma Species in Industrial Wastewater: Morphological and Molecular Insights from Isolates.

The genus Trichoderma holds economic significance due to its widespread distribution and diverse applications, including biological control, enzyme production, and various biotechnological uses. The accurate identification of Trichoderma species is crucial given their close association with human activities. Despite previous efforts in classification, a comprehensive analysis combining morphological and molecular approaches is necessary. This study focuses on the isolation of four Trichoderma species from industrial wastewater in Pakistan, expanding on the known diversity in the region; isolation involved collecting samples from industrial wastewater effluents at specific sites in Punjab, Pakistan. Trichoderma strains were cultured and purified on solid media, with subsequent biomass production for bisorptional activity. Morphological characterization included colony features and microscopic examinations. DNA extraction, polymerase chain reaction (PCR), and sequencing of the internal transcribed spacer (ITS) region were conducted for molecular analysis. Phylogenetic analysis was performed using the Maximum Likelihood Algorithm. The study identified three Trichoderma species, viz. T. citrinoviride, T. erinaceum, and T. longibrachiatum. Each species was characterized morphologically and supported by molecular-phylogenetic analysis. Illustrations of microscopic features and a phylogenetic tree based on the ITS-nrDNA region were recorded. T. citrinoviride and T. longibrachiatum, isolated from steel mill and tanneries wastewater, respectively, were differentiated based on morphological characteristics such as phialides and conidia. The combination of morphological and molecular techniques enhances the accuracy of species identification. The study highlights the significance of Trichoderma in industrial wastewater environments and underscores the need for continued research in this area. Future research should focus on exploring the ecological roles and potential applications of the newly identified Trichoderma species. Additionally, further investigations into the biotechnological potential of these species, including enzyme production and bioremediation capabilities, would contribute to their practical applications.

Phytochemical composition and antitumor activity of a new arctic lichen Anamylopsora pakistanica.

For the first-time, chemical composition and in vitro antitumor activity was investigated of a newly described lichen Anamylopsora pakistanica Usman & Khalid from the second highest plateau of the world (Deosai Plains, Pakistan). HPLC-UV method was used for identification of secondary metabolites and the acetone extract had higher values of TPC (41.90 mg GA/g) and TFC (75.37 mg RE/g) as compared to methanol extract. As chemical constituents 5,7-dihydroxy-6-methylphthalide, haematommic acid and alectorialic acid, were identified as major compounds. Atranol, alectorialin, gyrophoric acid and usnic acid were detected as minor substances. Acetone and methanol extracts induced a dose-dependent and time-dependent decrease in the viability of three types of tumour cells HeLa, HCT116 and MDA-MB-231. This lichen extract can induce S phase arrest in HeLa as compared to the untreated cells. Extract of this unique lichen, A. pakistanica, can be used safely as a significant source of biologically active compounds.

Two novel species of arctic-alpine lichen-forming fungi (Ascomycota, Megasporaceae) from the Deosai Plains, Pakistan.

Members of the lichen-forming fungal genus Oxneriaria are known to occur in cold polar and high altitudinal environments. Two new species, Oxneriariacrittendenii and O.deosaiensis, are now described from the high altitude Deosai Plains, Pakistan, based on phenotypic, multigene phylogenetic and chemical evidence. Phenotypically, O.crittendenii is characterised by orbicular light-brown thalli 1.5-5 cm across, spot tests (K, C, KC) negative, apothecia pruinose, hymenium initially blue then dark orange in response to Lugol's solution. Oxneriariadeosaiensis is characterised by irregular areolate grey thalli 1.5-2 cm across, K test (light brown), KC test (dark brown), apothecia epruinose, hymenium initially blue then dark blue in response to Lugol's solution. Both species share the same characters of thalli with black margins and polarilocular ascospores. The closest previously reported species, O.pruinosa, differs from O.crittendenii and O.deosaiensis in having non-lobate margins, thin thalline exciple (45-80 µm thick), short asci (55-80 × 25-42 µm) and K positive (yellow) and KC negative tests and divergent DNA sequence in the ITS, LSU and mtSSU regions. The newly-described Oxneriaria species add to growing evidence of the Deosai Plains as a region of important arctic-alpine biodiversity.

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.

DNA barcoding and scanning electron microscopy reveals Erysiphe ahmadii sp. nov. and a new record Erysiphe populicola (Erysiphaceae, Helotiales) on salicaceous hosts from Pakistan.

A new species of powdery mildew fungus Erysiphe ahmadii and a new record, Erysiphe populicola, on Salicaceae are described from Pakistan. In addition to light microscopy, scanning electron microscopy is also done to clearly demonstrate the surface characters of chasmothecia. E. ahmadii sp. nov. is characterized by large conidia ((-26)29-35(-37) × (-16)17-21(-23) µm), long chasmothecial appendages (198-286 µm) and small conidiophores. The novelty is confirmed by analyzing the genetic variation of internal transcribed spacer region (ITS1-5.8S-ITS2) of the ribosomal DNA gene, a universal fungal marker. E. populicola is characterized for the first time using molecular phylogenetic markers. Detailed descriptions along with scanning electron microscopy (SEM) photographs are provided in this paper. RESEARCH HIGHLIGHTS: Powdery mildews are obligate biotrophic pathogens of plants. Erysiphe ahmadii, a new powdery mildew fungus on willow trees, is described. First reference sequence of Erysiphe populicola is also generated. Both taxa are discussed in detail using macro- and micro-morphological and DNA barcoding techniques.

Connecting the multiple dimensions of global soil fungal diversity.

How the multiple facets of soil fungal diversity vary worldwide remains virtually unknown, hindering the management of this essential species-rich group. By sequencing high-resolution DNA markers in over 4000 topsoil samples from natural and human-altered ecosystems across all continents, we illustrate the distributions and drivers of different levels of taxonomic and phylogenetic diversity of fungi and their ecological groups. We show the impact of precipitation and temperature interactions on local fungal species richness (alpha diversity) across different climates. Our findings reveal how temperature drives fungal compositional turnover (beta diversity) and phylogenetic diversity, linking them with regional species richness (gamma diversity). We integrate fungi into the principles of global biodiversity distribution and present detailed maps for biodiversity conservation and modeling of global ecological processes.

First Report of Leaf Spot Caused by Corynespora cassiicola on Jasminum sambac in Pakistan.

Jasminum sambac L. is a species of jasmine native to a small region in the eastern Himalayas and is cultivated worldwide as an ornamental plant (USDA-ARS 2016). In Pakistan, it is cultivated for ornamental purposes throughout the country. The flowers of this plant are traditionally used in the preparation of essential oils and for making jasmine tea. The flowers and leaves also have been used in folk medicine to treat breast cancer, epilepsy, ulcers and promote wound healing (Al-Snafi 2018). In December, 2017, almost 10 leaves of 3 plants of J. sambac growing plant nursery of Gehlan, Pattoki, Punjab a province of Pakistan were observed with leaf spot disease. Infected leaves exhibited circular to sub-circular spots with indistinct margins and grey papery centers delimited by dark brown rims. For further microscopic study, the infected leaves were examined under a stereomicroscope. For the isolation and cultural studies of infecting fungus, infected parts of leaves were surface sterilized in 1% NaOCl for about 10 seconds, washed twice in sterilized distilled water, plated on potato dextrose agar (PDA) medium and incubated at 25°C for 4 days. Pure cultures were obtained having colonies of light to dark brown color. Conidia (n=20) were light brown to pale olivaceous brown, smooth, obclavate to cylindrical in shape, 99.5-118.5 µm in length and 12.5-15.0 µm in width, with mostly 3 to 14 pseudosepta. Conidiophores (n=20) were straight to slightly curved, unbranched, and pale to light brown in color. Based on the morphological characteristics of the colonies and conidiophores and conidia, the pathogen was identified as Corynespora cassiicola (Berk and M.A. Curtis) C.T. Wei. (Berkeley & Curtis 1968; Lu et al. 2021; Wei 1950). Genomic DNA was extracted following using modified CTAB method (Gardes and Bruns 1993) and internal transcribed spacer (ITS) region was amplified with ITS1 and ITS4 primers (White et al. 1990). The ITS sequence generated of about 553 bp and deposited in GenBank (accession no. MN954556), was found more than 99% similar to previously deposited sequences of C. cassiicola (GenBank accession nos. MN339671, EU364535, FJ852574, MK139711, EU131374) as verified through BLASTn and phylogenetic tree construction. A pathogenicity test was performed for fulfilling Koch'spostulates. Conidial suspension (105 conidia/ml) of the recovered isolate was sprayed on the 5 healthy leaves of 2-month-old seedling of J. sambac. Mock inoculated plants sprayed sterile distilled water were used as a control. The seedlings were covered with plastic bags to maintain high humidity at 24 to 28°C for a week. Identical disease symptoms to those observed in nursery plants were observed on the leaves of the inoculated plants in 7 days but not mock inoculated plants and results were reconfirmed. The reoccurred fungus was isolated from the diseased spots of the inoculated leaves to complete Koch's postulates and identified microscopically. A representative sample of leaves with lesions was deposited in the LAH herbarium, Department of Botany University of the Punjab, Pakistan (LAH35691). Previously, C. cassicola has been found infecting Jasminum mesnyi in China and Jasminum sp. in Florida (Alfieri et al. 1984; Zhang et al. 2018). The best of our knowledge, this is the first report of leaf spot caused by C. cassiicola on J. sambac in Pakistan. It will establish a foundation for future studies of management strategies for this plant disease caused by C. cassiicola.

New species of Mallocybe (Agaricales, Inocybaceae) from Pakistan, based on morphological and molecular evidence.

Within the family Inocybaceae, many species of Mallocybe have been reported, but there are only a few reports of this genus from Pakistan. In this study, six collections of Mallocybe were studied by morphological and phylogenetic methods. Phylogenetic analyses, based on sequence data from two different loci (ITS and LSU) using Maximum Likelihood and Maximum Parsimony methods, have been performed to infer species relationships within Mallocybe. Results indicated that these six collections encompass two new species of Mallocybe i.e. M.pakistanica and M.pinicola, from Pakistan. Their detailed morphological descriptions and illustrations are also provided. In addition, comparison with morphologically closely-related taxa is also discussed. Previously, only two species of this genus have been recorded from Pakistan and, with this addition, the total number of reported taxa of Mallocybe has been raised to four from Pakistan. A key to the described taxa of Mallocybe from Pakistan is also provided.

Identification and taxonomic position of a new Pseudoomphalina species from Pakistan based on light, scanning electron microscopy, and molecular analysis.

Khanspur, a small town located near Ayubia National Park, is a famous mountainous resort lying in Galyat area of Khyber Pakhtunkhwa. It is included in one of the richest biodiversity hotspots in the country. Despite of extensive previous studies, many new species are awaiting to be documented, including macrofungi. In this study, a new macrofungus Pseudoomphalina khanspurensis is analyzed by both light and scanning electron microscopy as well as DNA sequences based on nrITS and nrLSU regions. The sister species P. cokeri differs by its red to purple, dark to reddish brown, broadly convex to applanate pileus, purple blue to brownish stipe, and numerous cylindrical to lageniform cheilocystidia. HIGHLIGHTS: This is the first report of genus Pseudoomphalina from Pakistan and first study based on Scanning electron microscopy. These species have been described with detailed micro-morphological and molecular makers (nrITS and nrLSU). General distribution, ecology, diagnostic features and comparisons with alies have been provided. Graphical representation of DNA extraction and geographical locations of sampling sites are also illustrated. Softwares such as CIPRES Science Gateway Portal, MUSCLE, BioEdit, FigTree, Adobe illustrator and Adobe Photoshop are used in the current study. No member of this genus is already described from Pakistan.

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.

Laccaria murina and L. pumila from Pakistan, a study based on SEM and nrITS molecular marker.

Two species of the genus Laccaria, are described as new reports for Pakistan. Laccaria murina has been collected from a Himalayan moist temperate forest in Khanspur, KP, while L. pumila was found in the moist temperate forests of Kumrat Valley in Dir Upper, KP, and at higher altitudes of the Deosai plains of Gilgit-Baltistan, Pakistan. Phylogenetic analysis based on the nrITS region clustered the Pakistani species with L. murina sequences with other Asian collections and L. pumila from USA and Netherlands. SEM of basidiospores along with detailed micro-morphological data are provided. General distribution, habitat, ecology, and diagnostic features are also discussed. RESEARCH HIGHLIGHTS: It has new reports from Pakistan, studies based on light, scanning electron microscopy, and nrITS molecular markers. These species have been described with detailed micro-morphological and molecular phylogenetic analyses. General distribution, ecology, diagnostic features, and comparisons with closely related specimens have been provided. Graphical representation of DNA extraction and geographical locations of sampling sites are also illustrated (Figures 1 and 2). Very few members of this genus are already described from Pakistan.

Two new species of genus Leucoagaricus (Agaricaceae, Agaricales) from Pakistan.

The genus of basidiomycetous fungi, Leucoagaricus, occurs worldwide, from subtropical to boreal latitudes. Several collections of Leucoagaricus were made during mycological field trips conducted in different forests of Margalla, Pakistan. An integrative framework combining morphological and phylogenetic data was employed for their study. As a result, the two species La.margallensis and La.glareicolor are here described as new to science. Detailed macro- and micro-morphological descriptions, and a molecular phylogenetic reconstruction based on nrITS and LSU sequence data are provided and used to discriminate the new species from morphologically and phylogenetically close taxa. Whereas, our phylogenetic tree inference gave unequivocal support for the inclusion of these two species within the section Leucoagaricus.

Global patterns in endemicity and vulnerability of soil fungi.

Fungi are highly diverse organisms, which provide multiple ecosystem services. However, compared with charismatic animals and plants, the distribution patterns and conservation needs of fungi have been little explored. Here, we examined endemicity patterns, global change vulnerability and conservation priority areas for functional groups of soil fungi based on six global surveys using a high-resolution, long-read metabarcoding approach. We found that the endemicity of all fungi and most functional groups peaks in tropical habitats, including Amazonia, Yucatan, West-Central Africa, Sri Lanka, and New Caledonia, with a negligible island effect compared with plants and animals. We also found that fungi are predominantly vulnerable to drought, heat and land-cover change, particularly in dry tropical regions with high human population density. Fungal conservation areas of highest priority include herbaceous wetlands, tropical forests, and woodlands. We stress that more attention should be focused on the conservation of fungi, especially root symbiotic arbuscular mycorrhizal and ectomycorrhizal fungi in tropical regions as well as unicellular early-diverging groups and macrofungi in general. Given the low overlap between the endemicity of fungi and macroorganisms, but high conservation needs in both groups, detailed analyses on distribution and conservation requirements are warranted for other microorganisms and soil organisms.

Diversity and ethnomycological importance of mushrooms from Western Himalayas, Kashmir.

BACKGROUND: Wild edible mushrooms (WEM) are economically significant and used in traditional medicines worldwide. The region of Jammu and Kashmir (Western Himalayas) is enriched with the diversity of edible mushrooms, collected by the rural people for food and income generation. This is the first detailed study on diversity and ethno-medicinal uses of mushrooms from the State of Jammu and Kashmir. METHODS: Consecutive surveys were conducted to record ethnomycological diversity and socio-economic importance of wild edible mushrooms value chain in rural areas of Azad Jammu and Kashmir during 2015-2019. Ethnomycological data were collected with a semi-structured questionnaire having a set of questions on indigenous mycological knowledge and collection and retailing of wild edible mushrooms. A total of 923 informants from the study area provided the results identifying the gender, type of mushroom species, medicinal uses, and marketing of mushrooms. Diversity of mushrooms was studied by using quadrat and transect methods. Principal component analysis (PCA) and detrended correspondence analysis (DCA) were also applied to the dataset to analyse the relationship between species distribution, the underlying environmental factors, and habitat types. PCA identified the major species-specific to the sites and put them close to the sites of distribution. RESULTS: A total of 131 mushroom species were collected and identified during 2015-2019 from the study area. Ninety-seven species of mushrooms were reported new to the State of Azad Jammu and Kashmir. The dominant mushroom family was Russulaceae with 23 species followed by Agaricaceae, 16 species. Major mushroom species identified and grouped by the PCA were Coprinus comatus, Lactarius sanguifluus, Amanita fulva, Armillaria gallica, Lycoperdon perlatum, Lycoperdon pyriforme, and Russula creminicolor. Sparassis crispa, Pleurotus sp, and Laetiporus sulphureus were recorded most edible and medicinally significant fungi. Morels were also expensive and medicinally important among all harvested macro-fungal species. These were reported to use against common ailments and various health problems. CONCLUSIONS: Collection and retailing of WEM contribute to improving the socio-economic status, providing alternative employment and food security to rural people of the area. These mushrooms are used as a source of food and traditional medicines among the rural informants and could be used as a potential source of antibacterial and anticancer drugs in the future.

A compendium of macrofungi of Pakistan by ecoregions.

Macrofungi form fruiting bodies that can be detected with the naked eye in the field and handled by hand. They mostly consist of basidiomycetes, but also include some ascomycetes. Mycology in Pakistan is still in its infancy, but there have been many historical reports and checklists of macrofungi occurrence from its 15 ecoregions, which range from Himalayan alpine grasslands and subtropical pine forests to deserts and xeric shrublands. In this work, we searched and reviewed the historical literature and the GenBank database for compiling a comprehensive list of macrofungi reported from Pakistan to date. We recorded 1,293 species belonging to 411 genera, 115 families and 24 orders. These occurrences were updated taxonomically following the classification system currently proposed in the Index Fungorum website. The highest represented order by taxon number is Agaricales (47%) with 31 families, 146 genera and 602 species, followed by Polyporales (11%), Russulales (9%) and Pezizales (8%). Genera occurrence reported therein are presented for each ecoregion to the best of our ability given the data. We also discussed the currently known macrofungi diversity between different ecoregions in Pakistan. Overall, this work should serve as a solid foundation for the inclusion of Pakistan macrofungi in global biodiversity and conservation studies.

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.

Description of the Fifth New Species of Russula subsect. Maculatinae from Pakistan Indicates Local Diversity Hotspot of Ectomycorrhizal Fungi in Southwestern Himalayas.

Russula subsect. Maculatinae is morphologically and phylogenetically well-defined lineage of ectomycorrhizal fungi associated with arctic, boreal, temperate and Mediterranean habitats of Northern Hemisphere. Based on phylogenetic distance among species, it seems that this group diversified relatively recently. Russula ayubiana sp. nov., described in this study, is the fifth in the group known from relatively small area of northern Pakistan situated in southwestern Himalayas. This is the highest known number of agaric lineage members from a single area in the world. This study uses available data about phylogeny, ecology, and climate to trace phylogenetic origin and ecological preferences of Maculatinae in southwestern Himalayas. Our results suggest that the area has been recently colonised by Maculatinae members migrating from various geographical areas and adapting to local conditions. We also discuss the perspectives and obstacles in research of biogeography and ecology, and we propose improvements that would facilitate the integration of ecological and biogeographical metadata from the future taxonomic studies of fungi in the region.

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.

First Report of the Rust Disease Caused by Puccinia crepidis-japonicae on New Host Sonchus arvensis from Pakistan.

Sonchus arvensis (Asteraceae) is a traditional medicinal herb. The aerial parts are a rich source of vitamins, essential amino acids, and minerals, which may help in treatment of fever, inflammation, detoxication, and blood circulation (Li et al. 2018). In October 2018, typical rust symptoms were observed on S. arvensis leaves and stems in Buner district, Malakand division, Khyber PakhtunKhwa Province, Pakistan (34.39°N; 72.61°E). Almost 40% of leaves and stems of five S. arvensis plants displayed severe rust. The specimens were examined microscopically and compared with type specimen description in published literature (Dietel 1908; Hiratsuka et al. 1992). The fungus was identified as Puccinia crepidis-japonicae based on the characteristic of spore morphology and phylogenetic study based on the internal transcribed spacer (ITS) and large subunit (LSU) sequence data. Uredinia were amphigenous, rounded, or somewhat elliptical, naked, small patches, equally spread, brown. Urediniospores (n=30) were globose to ellipsoid, brownish yellow and measured 22.4-24.7 × 20.2-22.1 µm. Urediniospore walls were brownish orange and finely echinulate and 1.7 to 2.1 µm thick with 2 to 3 germ pores. Telia were amphigenous, rounded or elliptic, scattered, dark brown to blackish. Teliospores (n=30) were ellipsoid, subglobose or long ellipsoid, rounded at both ends, not thickened at apex, warted, reddish brown, and measured 31.3-39 × 24.6-26.8 µm. Teliospore walls were reddish-black and about 1.5-2.5 µm thick, and the pedicles were short, hyaline, fragile, become tapered toward apex, and measured 14.4-18.7 × 4.7-9 µm. DNA was extracted from urediniospores, and the combined region of ITS and LSU (28S) were amplified using Rust2Inv (forward primer) and LR6 (Reverse primer) according to the protocol outlined by Aime (2006). A BLASTn search (http://www.ncbi.nlm.nih.gov) showed that the combined ITS and LSU region shared 99% identity (792/804 bp) to the P. crepidis-japonicae accessions (KY798395 from Hawaii, USA) with 100% query cover. The resulting sequence was deposited in GenBank (Accession No. MN093335). Both morphological and molecular characteristics indicatethat this species was P. crepidis-japonicae. To test pathogenicity and fulfill the Koch's postulates, a urediniospore suspension (1 × 104 spores/ml) was sprayed on three 6-week-old plants of S. arvensis, and one as negative control, incubated at 22-24°C. Uredinia were observed on the leaves after 10 days of inoculation, whereas the control plants remained symptomless. Microscopic examination confirmed that the symptoms on plants obtained from the field and greenhouse inoculations were morphologically identical. This fungus has been observed previously on Crepis japonica in China, Hong Kong, Japan, Korea, and Taiwan, on Prenanthes spp. in China and on Youngia tenuifolia and Y. fusca in China (Farr and Rossman 2021). Pereira et al. (2002) suggested that P. crepidis-japonicae may play a significant role as a biocontrol agent against its weed host. To the best of our knowledge, there are no other reports of this fungus on any other hosts in Pakistan. The specimen has been vouchered in LAH Herbarium, Department of Botany, University of the Punjab, Lahore, Pakistan (LAH36343). This is the first report of P. crepidis-japonicae on S. arvensis as a new host from Pakistan.