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Title:   Author  item
                                            Lartey, Robertitem
                                            Caesar, Thecanitem
                                            Caesar, Anthonyitem
                                            Sainju, Upendraitem
                                            Evans, Robert
  Submitted to: Plant Disease
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 8/24/2012
                                    Publication  Date: 1/1/2013
                                    Citation: Lartey, R.T., Caesar, T., Caesar, A.J., Sainju, U.M., Evans, R.G. 2013. First report of spot form net blotch caused by Pyrenophora teres f. maculata on barley in the Mon-Dak area of the US. Plant Disease. 97(1): 143-143.Interpretive Summary: The common foliar disease, net blotch of barley is caused by the fungus Pyrenophora teres around the world. Two forms of the disease are recognized. These are net form net blotch (NFNB) caused by P. teres f. teres and a spot form net blotch (SFNB) caused by P. teres f. maculata. In the Mon-Dak area, as in other parts of the Northern Great Plains, NFNB is widespread. SFNB has been reported in Western Montana and more recently in Eastern North Dakota but not in the Mon-Dak area. In the summer of 2011, some spot lesions that were surrounded by necrosis or chlorosis were observed on different barley crops in fields at Williston ND, Nesson Valley ND and Sidney MT areas. This observation led to morphological and conidial suspensions examination for occurrence of SFNB in the area. The result did not show significant differences from control P. teres f. teres which is consistent when compared to that of P. teres f. maculata.  Pathogenicity test was then carried our on barley cultivar Tradition plants using two fungal isolates from each location.  Regardless of source the pathogen, spot lesions surrounded by necrosis or chlorosis, typical of SFNB, appeared on the inoculated leaves within 7 days. Leaves from these spot-form lesions were harvested and subjected to conidial examination. The fungi isolated from these lesions were again identified as P. teres based on the morphology of the conidia. All control plants which were sprayed with sterile distilled water were symptomless. Finally, direct PCR was carried out on of the internal transcribed spacer (ITS) region of ribosomal genes on both field and pathogenicity test leaf lesion samples to confirm P. teres f. maculata detection. The PCR products were sequenced and the sequences were compared to controls and samples from GenBank. The comparisons finally confirmed the presences of P. teres f. maculata from both sample lesions from the three locations and the pathogenicity tests. Thus, confirming for the first time the occurrence of SFNB in the Mon-Dak area.
                                Technical Abstract: Pyrenophora teres Drechs. causes net blotch of barley, a common foliar disease in cultivation zones around the world. The disease occurs in two forms, namely a net form net blotch (NFNB) caused by P. teres f. teres and a spot form net blotch (SFNB) caused by P. teres f. maculata. As in other parts of the Northern Great Plains, in the Mon-Dak area (Western North Dakota and Eastern Montana) NFNB is prevalent. SFNB was first reported in Western Montana in 1983 (1) and more recently in Eastern North Dakota in 2010 (3) but not in the Mon-Dak area. In the summer of 2011, unusual spot lesions that were surrounded by necrosis or chlorosis were observed on different barley cultivars (cvs.) in fields at Williston ND, Nesson Valley ND and Sidney MT areas. This observation led to an examination for occurrence of SFNB in the area, by transferring diseased leaves from various barley cvs. from the three locations to water agar and incubating them at room temperature for 24 hrs to induce sporulation. Morphological examination of conidial suspensions (45-169 x 15-21 μm) did not show significant differences from control P. teres f. teres (provided by Tim Friesen, ARS, Fargo, ND). For pathogenicity testing, six 14 day-old plants of barley cultivar Tradition were sprayed until runoff with a 2000 spore/ml suspension of two isolates from each location and the control, incubated first in a lighted humidity chamber for 24 h and then in a greenhouse for seven days at 21°C. Regardless of inoculum source, spot lesions surrounded by necrosis or chlorosis, typical of SFNB, appeared on the inoculated leaves within 7 days. Leaves showing spot-form lesions were harvested and subjected to conidial examination. Fungi isolated from the leaves were identified as P. teres and the morphology of the conidia was undistinguishable from those of P. teres f. teres. All control plants which were sprayed with sterile distilled water were symptomless. Two independent inoculations tests were performed. Direct PCR (2) of the internal transcribed spacer (ITS) region of ribosomal genes was performed on field and pathogenicity test leaf lesion samples to confirm P. teres f. maculata detection. PCR templates were prepared using the Extract-N-Amp Plant PCR Kits (Sigma Chemical Co. St. Louis, MO) and subjected to PCR using ITS1 and ITS4 primers. Amplicons were sequenced as previously described (2). The nucleotide sequences of P. teres f. maculata from MonDak area were submitted to GenBank under Acc nos: PtmNES1 (JX187587), PtmSDY1 (JX187588), PtmSDY2 (JX187589) and PtmWIL1 (JX187590). Sequences aligned (100 % similarity) with GenBank sequence, P. teres f. maculata (EF452471) while showing 10 nucleotide differences (99% similarity) with P. teres f. teres (EF452472). Related sequences from other common pathogens were retrieved from GenBank included Cochliobolus sativus (JN943406 and JQ068818), P. tritici-repentis (AY739796 and F452491) Rhynchosporium secalis (AF384680 and AY140668).  These were compared to our test sequences by alignment and a neighbor-joining phylogenetic tree was constructed using ClustalW2 in the Vector NTI software package (ver 11). All of the test sequences clustered with those from P. teres f. maculata while separating the other species on distinct tree branches (not shown). The results confirm first report of SFNB in the Mon-Dak area.


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