A specific and sensitive PCR assay for the detection of Phytophthora infestans , the cause of late blight of potato, in soil and plant tissues was developed. A P. infestans -specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of rDNA internal transcribed spacer regions of most of the known Phytophthora species. PCR amplification of P. infestans DNA with primers INF FW2 and INF REV generated a 613 bp product, and species specificity was demonstrated against DNA from nine other Phytophthora species and seven potato-blemish pathogens. In a single-round PCR assay, 0·5 pg pure P. infestans DNA was detectable. Sensitivity was increased to 5 fg DNA in a nested PCR assay using Peronsporales-specific-primers in the first round. As few as two sporangia or four zoospores of P. infestans could be detected using the nested assay. Procedures are described for detection of P. infestans in leaves, stem and seed potato tubers before expression of symptoms. A soil assay in which 10 oospores per 0·5 g soil were detectable was developed and validated using samples of field soil. The PCR assay was used to examine the long-term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of P. infestans in leaf material buried in a replicated experiment under natural field conditions. Oospores were consistently detected using the PCR assay up to 24 months (total length of the study) after burial in soil, whereas the sporangial inoculum was detected for only 12 months after burial. Sporangial inoculum was shown to be nonviable using a baiting assay, whereas leaf material containing oospores remained viable up to 24 months after burial.

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C otton has vital role in economy of Pakistan. It is main source of fiber as well as vegetable oil. It is major source of foreign exchange obtained by exporting raw cotton to many countries of world. Cotton production and area is continuously reducing in Pakistan due to biotic and abiotic stresses and competition from other crops.

Rice (Oryza sativa L.) is one of the highly consumed cereal grain crops in Pakistan. In September 2017, leaf samples of cultivar Basmati-385 showing brown to dark brown spots (5 to 9 mm in diameter) that were oval or cylindrical in shape with a chlorotic yellow halo and grayish tan centers were collected from fields near the University of Agriculture, Faisalabad (31.43633 N 73.05981 E). Average disease incidence was 69% in six rice fields that were sampled for diseased plants with visible symptoms. To isolate the pathogen, from 20 diseased leaves, 5 mm2 segments from the margins of lesions were cut, rinsed with sterile distilled water (SDW), surface disinfected by 70% ethanol and again rinsed with SDW. The samples were dried on sterilized filter paper discs, plated on potato dextrose agar (PDA) and incubated at 27°C for 5 to 7 days. Twelve isolates were sub-cultured and single-sporing was performed to obtain pure cultures. Fungal isolates with light to dark gray in color, thick or fluffy aerial mycelium, circular and smooth margins were obtained after 7 days of incubation. Conidia were 47-83 μm × 10-17 μm (n=100), with 4 to 10 distosepta, dark or olivaceous brown, straight or moderately curved, and the cells at the ends occasionally looked paler than those in the middle. Conidiophore of the fungus were simple, smooth, cylindrical, septate, and straight to flexuous. These characteristics resembled those of Bipolaris zeicola (Stout) Shoemaker (Manamgoda et al. 2014). For molecular identification, genomic DNA (isolate SU-11) was extracted and the internal transcribed spacer (ITS) region, large subunit (LSU) of ribosomal DNA, translation elongation factor (tef), glyceraldehyde 3-phosphate dehydrogenase (gpd), and RNA polymerase II second largest subunit (rpb2) genes were amplified and sequenced by using the primers ITS1-F/ITS4-R (White et al. 1990), LROR-F/LR5-R (Schoch et al. 2012), EF1-983F/EF1-2218R (Rehner and Buckley 2005), GPD1F/GPD2R (Berbee et al. 1999), and 5F2/7CR (O’Donnell et al. 2007), respectively. BLASTn searches showed 100% homology with the LSU and rpb2 sequences of B. zeicola (GenBank Accession Nos. MH876201 and HF934842) and 98-99% similarity with ITS, tef, and gpd sequences of B. zeicola (GenBank Accession Nos. KM230398, KM093752 and KM034815). The sequences of ITS, LSU, tef, gpd, and rpb2 were deposited in GenBank with accession numbers MN871712, MN877767, MN867685, MN904511 and MT349837, respectively. To fulfill Koch’s postulates, 25 greenhouse-grown rice plants (cv. Basmati-385) at 2- to 3-leaf stage were spray inoculated with a spore suspension (105 spores/ml; isolate SU-11) prepared in SDW. Plants were covered with plastic wraps to maintain humid conditions for 24 hours and incubated at 27°C for one week. Similarly, ten non-inoculated plants sprayed with SDW served as controls. After one week, observed symptoms were similar to those from natural infections and no disease symptoms were observed on the non-inoculated plants. The experiment was repeated twice and the pathogen was re-isolated from the infected leaves and characterized morphologically. Globally, B. zeicola has also been reported to cause the leaf spot of rice and maize plants (Sivanesan 1987; Kang et al. 2018). To our information, this is the first report of B. zeicola causing brown leaf spot of rice in Pakistan. The increasing risk of this fungal pathogen in the rice-growing areas of Pakistan need a rigorous exploration and outreach effort to develop effective management practices.

Wheat [Triticum aestivum L. (Poaceae)] is the staple diet of people in Pakistan. It is attacked by many types of pests. Therefore the purpose of this study was to assess the impact of climate change on the ecology and epidemiology of various wheat pests in Punjab, Pakistan. Results indicate that maximum weeds hot spots 242 (5.98%) Phalaris minor, 45 (1.18%) wild oat and 203(5.01%) broad leaf weeds were noted in 2015. Aphid 31 (0.86%) hot spots were recorded in 2016 while maximum army worm 13 (0.26%) hot spots were noted in 2017. Maximum 70 (1.73%) spots of yellow rust and 85 (2.10%) hot spots of brown rust were observed during 2015 while 84 (4.16%) spots of loose smut were observed during 2017. ANOVA shows that years have no significant difference (P > 0.05) but weeks have significant effect on occurrence of these pest incidences except brown rust. But regression ANOVA was significant (P ≤ 0.05) and regression models equations have been developed on the bases of recorded data. Pest incidence was taken as dependent variable Y and weather factors i.e. minimum temperature as X 1 , maximum temperature as X 2 , relative humidity as X 3 and rain fall as X 4 were taken as independent variables. This study will help in recommendations for moving forward aiming at integration of biology of rust and smut diseases of wheat with changing climate for development of resistant varieties for resilient and durable management of these pathogens.