During a field survey of greenhouses and fresh markets in 2013, fruits of tomato, oranges, and apples exhibited rot symptoms with white mycelial growth and salmon-color sporulation in the vicinity of Sargodha city (32°5′1″ N, 72°40′16″ E), Pakistan. Diseased fruit samples were collected in plastic bags and taken to laboratory on ice for further diagnosis. Diseased fruits were observed under a stereo microscope and single spores were removed using an inoculating needle. Isolation from single spores showed pink to white colonies on potato dextrose agar (PDA) containing hyaline, 2-celled, ellipsoid to pyriform conidia (17 to 24 × 7 to 11 μm) with slanting and truncate basal mark and produced in clusters. Conidiophores were branched (105 to 254 × 2 to 4 μm) and hyphae were hyaline (3 to 5 μm in diameter). These characteristics of the fungus were similar to Trichothecium roseum (Pers.) as reported by Inácio et al. (1). Genomic DNA was extracted by using CTAB buffer from a single pure colony of one isolate of the fungus and PCR analysis was performed for ITS region and part of the 5′ end of the beta tubulin (TUB) gene (2,3). Single fragments of 550 bp and 1.5 kb length from ITS and TUB gene were amplified and sequenced (GenBank Accession Nos. KF975702 and KJ607590, respectively). Sequence analysis showed 99% similarity with T. roseum isolates from different regions of the world. Phylogenetic analysis (MEGA version 5.2 with WAG model) showed the close relatedness to the isolates of T. roseum from Pakistan and isolates from other parts of the world that revealed the low genetic variability of ITS region. TUB gene sequence analysis indicated 100% homology with isolates of T. roseum and to the other species in Hypocreales. Pathogenicity tests were performed on tomato cvs. Nova Mech and Rio Grande, orange cv. Kinnow, and on apple cv. Golden Delicious by inoculating five fruits from each cultivar. Spore suspensions (105 conidia/ml of sterilized distilled water) were inoculated into all wounded fruits (9 wounds/fruit) of each cultivar and incubated at 25°C for the development of symptoms. Five wounded fruits of each cultivar were inoculated with sterilized distilled water as a control treatment. The fruits were kept in plastic boxes and incubated in humid chambers for 5 days. The symptoms on apples were observed as brown rot with pinkish spores on rotted tissue. The cross section of apple fruits also showed the brown rotted tissues internally. The fungus developed mycelium and spores on the surface and caused severe rotting inside the tomato and citrus fruits. T. roseum was re-isolated by picking a single spore from rotted tissues of fruits under a stereo microscope, and culturing on PDA. The re-isolated fungus was confirmed morphologically and by molecular techniques. Tomato and apple has been reported as a host for T. roseum (1,4,5) but oranges have not. To our knowledge, this is the first record of T. roseum infecting tomato, oranges, and apples in Pakistan. References: (1) C. A. Inácio et al. Plant Dis. 95:1318. 2011. (2) K. O'Donnell, and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (4) Y. H. Yun et al. Afr. J. Microbiol. Res. 7:1128, 2013. (5) M. Žabka et al. Mycopathologia. 162:65, 2006.
Identification of Trichoderma Isolates from Tobacco Growing Regions of West Godavari District, Based on Sequence Analysis of ITS Region of rDNA and Morphological Variations in the Strains
