Archivio Journal of Plant Pathology

Abstract:

In August 2014, seeds of Glycine max in storage rooms at Lahore, Pakistan, were found to be colonized by an unknown fungus. For pathogen isolation, seeds were incubated on moist blotters, developing a green mass of fungal spores. Spores were transferred to Czapek Dox agar medium and incubated at 25±2°C. After 7 days of incubation colonies were velvety, green and floccose, consisting of white vegetative mycelium and acquired the diameter of 3-4 cm. Globose dark brown sclerotia were produced, smaller than A. flavus. Radiate conidial heads were observed that were mostly biseriate however uniseriate heads were also present. Conidiophores were hyaline, coarsely roughened and 0.9-1.2 mm in length. Vesicles were subglobose to globose, 25-40 μm in diameter, while metulae were 8-12 μm and phialides 5-8 μm long. Conidia were pale or olive green, 3.5-5 μm in diameter. Pathogen was identified as Aspergillus minisclerotigenes (FCBP1353) (Pildain et al., 2008) and differentiated from A. flavus and A. parvisclerotigenus by studying aflatoxin profiles (B1, B2, G1, and G2) using thin-layer chromatography. Amplification of DNA fragment of 650 bp was done using the universal primers ITS1/ITS4 (Batista et al., 2008) and total genomic DNA as a template (White et al., 1990). BLAST analysis of this strain (GenBank accession No. KJ564033) showed 99% similarity to different strains (JX292091, JF412776, KF841549, JF412775). Phylogenetic analysis of identified pathogen and closely related species of A. flavus group by the Maximum Likelihood Hood tree method and profiling of DNA bands examined in the members of Aspergillus section Flavi amplified by three different Inter Simple Sequence Repeat (ISSR) primers clearly confirmed the pathogen identity. Surface sterilized healthy seeds were soaked in spore suspension (104 spores ml-1) from a one week old pathogen culture for 30 s, dried and transferred onto moist blotting paper in Petri plates. Control seeds were treated with sterilized distilled water. After 7 days incubation at 25°C, 90% of seeds were colonized and re-isolation of the same pathogen confirmed the Koch's postulates. To our knowledge, this is the first report of A. minisclerotigenes seed rot from Pakistan.