Gulshan Irshad

First report of postharvest rot caused by Pestalotiopsis sp. on grapes in Punjab, Pakistan

Grapes (Vitis vinifera L.), a major crop in Pakistan, are cultivated mostly for the fresh market. In June 2016, severe fruit rot was observed in packing houses in Rawalpindi (33°38′19.2″N, 73°01′45.0″E) and Chakwal (32°56′01.5″N, 72°50′37.1″E) districts of Punjab province on cvs. King’s Ruby and Perlette after 3 to 5 days of postharvest storage. To determine incidence of symptoms, 10 boxes of each grape variety were selected randomly from each of five packing houses per district. Disease incidence was recorded at 4 to 6%. Initial symptoms occurred mainly on wounded berries; following infection, the fruit skin turned reddish brown, after which the area of discolored skin increased and whitish mycelium developed on the lesions. A total of 400 symptomatic berries were surface sterilized by immersing cheese cloth bags containing detached berries in 1% sodium hypochlorite for 1 min, then rinsing three times with sterile distilled water and allowing the berries to dry by placing them on filter paper for 45 s. Surface sterilized tissues at the margins of lesions were excised into 4-mm³ segments and then transferred to potato dextrose agar (PDA) and incubated at 25 ± 2°C. After 1 week, fungal colonies were white, floccose, and cottony, with black acervuli forming distinct concentric rings. A total of 29 isolates were examined morphologically; conidia were fusiform, straight or slightly curved, 4 to 5 celled, and 19.7 to 30.2 × 4.8 to 6.2 μm. The color of conidia was dark umber to olivaceous with a darker band at the septum between cells. Two to three appendages (22.6 ± 0.6 μm long) appeared at the apex of each conidium; apical appendages were not knobbed, whereas the basal end had a single appendage with a knobbed tip that was 5.7 ± 0.4 μm long. According to colony and conidia morphology, the isolates were identified as Pestalotiopsis clavispora (G.F. Atk.) Steyaert (Gonzalez et al. 2012; Guba 1961). The internal transcribed spacer (ITS1, 5.8s, and ITS2) region of isolates PSTL01, PSTL06, and PSTL12 was amplified using ITS1 and ITS4 primers. Sequence comparison revealed 99% genetic homology (accession nos. MF448223, MF571908, and MF571909) with previously reported isolates of P. clavispora (KY319134 and KM402033). To confirm pathogenicity, 10-µl aliquots of conidial suspension (10⁶ spores/ml) of PSTL01, PSTL06, or PSTL12 were pipetted onto three nonwounded and four wounded asymptomatic grape berries of cv. King’s Ruby per isolate; sterile distilled water was used as a negative control. The berries were incubated at 25 ± 2°C in sterile moist chambers, and the experiment was conducted twice. After 3 days, whitish mycelium and sunken lesions, similar in appearance to the original lesions, developed on both wounded and nonwounded inoculated berries, whereas no symptoms appeared on the negative controls. The morphology of the fungus that was reisolated on PDA was identical to that of the original cultures. Pestalotiopsis sp. has been reported to cause postharvest fruit rot on grapes in Korea (Deng et al. 2013). To our knowledge, this is the first report of Pestalotiopsis sp. causing fruit rot of grapes in Pakistan, where the disease poses a significant threat to sustainability of grape producers.

FIRST REPORT OF CURVULARIA LUNATA CAUSING LEAF SPOTS ON LOQUAT FROM PAKISTAN

The loquat (Eriobotrya japonica Lindl.) is achieving significant value in Pakistan. During the year 2014-2015, reddish brown circular spots were observed on leaves; these spots increased in size and coalesced to form oblong lesions. Samples were cut into small pieces (3 mm2), surface sterilized with sodium hypochlorite and transferred on Malt Extract Agar (MEA). These samples were incubated at 24±2°C and dark brown colonies with large stroma were observed. Septate condiophores were unbranched or simple and geniculate with dark brown scar. Brown conidia were straight to pyriform with 3-4 cells (large and curved center cell), ranging in size from 6.9 to 20.99 μm and produced apically in a sympodial manner. Morphological characterization was similar to Curvularia lunata (Ellis, 1971). PCR was used for amplification of rDNA region (White et al., 1990) and sequences of PAK34 and PAK35 was submitted to GenBank under accession Nos. KT280007 and KT280008. Both isolates were exhibiting 99% genetic homology with previously reported C. lunata isolates (KM246260, KM246262). Spore suspension (1 × 106 spore/ml) was sprayed on healthy loquat seedlings, kept in glasshouse at 30°C and plants developed lesions similar to those observed on naturally infected plants. Isolation was performed from inoculated loquat plants and morphological characterizations were similar to naturally isolated pathogens. Curvularia leaf spot diseases caused by C. lunata have been previously found on small evergreen tree. To our knowledge, this is the first report worldwide of C. lunata causing leaf spots on loquat.

Trinacrium anchorum, a new hyphomycetous fungus from Pakistan

Trinacrium anchorum sp. nov. is recorded from the branches of Vitis trifolia L., a member of the family Vitaceae. This plant was climbing over the building of Biological Sciences, Quaid-i-Azam University Islamabad, Pakistan. This fungus is different from all the previously described species, having bigger and definitely T-shaped conidia with gradually narrowing arms, bending down towards the main axis. The conidia of this species of Trinacrium are similar to an anchor; hence it is named ‘anchorum’.