First Report of a Ceratocystis manginecans Causing Quick Decline of Psidium guajava in Pakistan.

Psidium guajava is a widely grown fruit tree of Asia for food and medicinal purposes. Also being reported to have anti-inflammatory, antimicrobial, antioxidant, antidiarrheal, antimutagenic properties (Somu, 2012). In April 2018, quick decline disease of guava was observed in orchards of Sheikhupura, Lahore, Faisalabad, Kasur and Chiniot districts of Punjab, Pakistan. Approximately 68% of the trees were found declined with mummified fruits. Initial infection symptoms appeared as wilting of leaves, bark discoloration, followed by the leaf drooping, crown area discoloration, bark splitting, mummified fruits, dying of branches and lately whole tree death in weeks to months. The fungus formed a dark brown to black discoloration (3 to 5 cm wide and 7 to 9 cm long) in vascular bundles of P. guajava tree. Sixty-five samples of discolored wood from the main stem were collected, and pathogen was isolated using carrot bait method (Moller and DeVay, 1968). Isolation and purification were done on 2% Malt extract agar (MEA) plates incubated at 25 ± 2 °C in 12 h light and dark period. After 6 days of incubation, fungal hyphae, fruiting structures, sexual & asexual spores were observed on MEA plates. Black globose to subglobose ascomata with bases (151-) 200 (-278) µm in diameter with long neck (511-) 535 to 600 (-671) µm long, (23-) 28 to 39 (-47) µm wide at base, (13-) 13- 19 (-25) µm wide at tip and light brown to hyaline divergent ostiolar hyphae (50µm) were developed and produces hat-shaped hyaline ascospores 3 to 5 µm long and 6-7 µm (with sheath) and 4 µm (without sheath) wide. After 7 days, initially white mycelium turned into olivaceous green and produced primary phialidic conidiophore with emerging primary cylindrical hyaline conidia (7 to 12 × 4 to 6 µm), secondary conidiophore with emerging chain of secondary barrel-shaped hyaline conidia (9-) 10 to 12 (-13) µm long × (5-) 5 to 9 (-11) µm wide and dark brown dematiaceous chlamydospores conidia (12 ×10 µm) were observed. All morphological characteristics were consistent to the description of Ceratocystis manginecans (Van Wyk, et al., 2007). For further confirmation, from a purified isolate GWD10, genomic DNA was extracted. The internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF 1-α) region were amplified with primer pairs ITS1/ITS4 and EF1/EF2 (Jacobs et al., 2004; White et al., 1990) respectively. Generated sequences (Accession Nos. MN 365128 & MT952139) on BLAST analysis showed 100% homology for ITS and TEF with Ceratocystis manginecans (Accession No., KC261852 CMW 13582 Voucher, NR-119532.1 type material, MH863135; EF433317, respectively) reported from Oman and Pakistan (Van Wyk et al., 2007 & Vu et al., 2019). For pathogenicity test, one-year-old healthy P. guajava plants were inoculated by making a T-shaped slit of 5 × 7.5 mm in the bark. Two weeks old cultures of GWD10, 5-mm mycelial discs were aseptically transferred and covered with moistened sterilized cotton swab followed parafilm to maintain humidity. Fifteen plants were inoculated with fungal cultures and five plants were inoculated with MEA plugs as controls. All plants were maintained at 25 ± 2 °C with 80 ± 5% relative humidity (RH) in greenhouse Initial bark discoloration developed after 14 days of inoculation. After 40 days of inoculation plants started wilting and dying, similar to the symptoms were observed in naturally infected trees. Control plants remained asymptomatic. To fulfill Koch's pustulates, the same pathogen was re-isolated from the test plants and identified on morphological features to GWD10. The pathogen has been associated with mango decline in Oman and Pakistan (Van Wyk et al., 2007), acacia wilt in Indonesia (Harrington et al., 2015) and siris wilt in Pakistan (Razzaq et al., 2020). P guajava is an important fruit and medicinal plant, and the infection of C. manginecans is a great concern to the producers of P. guajava (Harrington et al., 2015; Huang et al., 2003). To our knowledge, this is the first report of Ceratocystis manginecans causing quick decline of P. guajava worldwide.

First report of Nigrospora oryzae causing wilt in summer cypress (Bassia scoparia) in Pakistan.

Summer Cypress (Bassia scoparia) is a large annual herb belonging to the family Amaranthaceae native to Eurasia. It has been introduced in many other countries of the world. In Pakistan, summer cypress is also known as kochia and grown as an ornamental plant for its red fall foliage for landscapes. During October, 2017 a survey was conducted in Punjab Province, Pakistan, where 100 wilted plant samples were collected from 30 different plantations of Faisalabad district. Up to 50% loss of plantation was noted in all visited locations. Lower parts of the plants were affected first presenting with necrosis of leaf tips surrounded by a chlorotic zone (Fig. 1. A). Then necrosis of apical margins of the plant parts occurred, followed by stem discoloration and wilting of entire herbaceous branches, leading to the partial wilting of the plants. Ultimately, whole plant wilted and died, (Fig. 1. B) appearing as though they had been scorched. Diseased tissues from lower stem (crown portion) were sampled, surface sterilized in 70 % ethanol for 30 s, and cultured on to Potato Dextrose Agar (PDA) medium. Petri dishes were incubated at 25 ˚C with alternating 12-hour periods of light and dark. Frequently observed, fast growing whitish grey fungal cultures with black pin head points were obtained after 7 days (Fig. 1. C). Young conidia were one-celled, yellow to orange in color and turned brown to black (Fig. 1. D & E), ranged in size from 11 µm to 16 µm x 9.5 µm to 12 µm (Fig. 1. F), and were ellipsoidal at maturity (Fig. 2. A). Hyphae were branched, septate and dark brown in color while conidiophores were flexuous, branched and ranged between 3.5 µm to 4.5 µm in diameter and 14.5 µm to 26.5 µm in length. Based on morphology (Ellis, 1971), the pathogen was identified as Nigrospora oryzae and submitted to the Westerdijk Collection of Fungi, Netherland (CBS 146145/RNOEG30). Total DNA of isolate EG30 was extracted and portions of the Internal transcribed spacer (ITS) region and beta-tubulin (ßt) gene were amplified using the universal primers ITS1F and ITS4 (White et al., 1990) and ßt2a and ßt2b (Glass and Donaldson, 1995). The generated ITS (GenBank Accession No. MG745331.1 491 bp) and ßt (GenBank Accession No. MN629896 408 bp) sequences were searched against GenBank using BLASTn and were 99% homologous to ITS (KX986074 525 bp ; MN341493 550 bp) and 100% homologous to ßt (MK262852 409 bp) gene region from Nigrospora oryzae (Wang et al., 2017; Zhang, 2019). For pathogenicity tests, ten healthy two-month-old summer cypress plants were inoculated by soil drenching of a spore suspension (106-107 spores/mL) of the fungal isolate EG30 while five plants were treated with sterilized water and used as control treatments. Plants were incubated at 60 to 75% relative humidity (RH) and 25 ˚C in a greenhouse. Leaf necrosis and partial to whole plant witling (Fig. 2. B & C) were observed in the inoculated plants after 21 days. No symptoms appeared in control plants. A fungus was re-isolated from the lower stem (crown portion) parts of the inoculated plants that was identical in morphology to isolate EG30. No fungus resembling EG30 was isolated from the control plants. To the best of our knowledge, this is the first report of summer cypress wilt caused by Nigrospora oryzae (Berk. and Broome) Petch, a known pathogen of several important crops in China, Australia, India, Canada, and Pakistan (Sharma et al., 2013).