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.

Termitomyces pakistanensis, a new mushroom species from Pakistan based on scanning electron microscopy and ITS-rDNA barcoding.

Termitomyces pakistanensis sp. nov. is a member of an edible genus generally distributed in Asia and Europe. This species has been described as new species based on its different morphology, and scanning electron microscopy (SEM) of basidiospores. The novelty and degree of endemism is confirmed by analyzing the genetic variation of the internal transcribed spacer regions (ITS1-5.8 S-ITS2) of the ribosomal DNA gene, a universal fungal marker. The evolutionary affinities of new species is also evaluated with Asian and European species by phylogenetic analysis based on ITS sequences. In our phylogenetic analysis, this genus is found monophyletic comprising of two monophyletic sub clades: Clade I, Microcarpus, with small sized fruiting bodies generally less than 5 cm without pseudorrhiza and Clade II, Macrocarpus, with large sized fruiting bodies generally more than 5 cm having pseudorrhiza. All collections of Pakistani species clustered independently in Microcarpus clade showing their endemic genetic makeup as it is clustering independently. A comprehensive description, photographs of the basidiocarps and Scanning electron microscopy (SEM) micrographs of spores are provided. RESEARCH HIGHLIGHTS: It has a new species from Pakistan to world based on the scanning electron microscopy and further confirmed by DNA barcoding. The exact shape and size of basidiospores of this novel species is first time introduced by using SEM analysis. This genus is rarely described from Pakistan. This paper has introduced a two clade, Microcarpus and macrocarpus, in the world for this genus.

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.