First report of Alternaria alternata causing Alternaria leaf spot of Fig in Pakistan.

Fig (Ficus carica) is a species of flowering plants within the mulberry family. During June 2020, leaf spots were observed on several fig plants (31°26'15.0"N 73°04'25.6"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, oval to circular, light brown, sunken spots that were uniformly distributed on the leaves. Spots gradually enlarged and coalesced into circular to irregular dark brown to black spots that could be up to 3cm diam. with no or small sized fruit. Disease incidence was approximately 25%. To identify the causal agent of the disease, 15 symptomatic leaves were collected. Small pieces from all diseased samples were removed from the margin between healthy and diseased tissues were surface disinfested in 70% ethanol for 2 min, rinsed three times with sterile distilled water, plated on Potato dextrose agar and incubated at 25 ± 2°C with a 12-h photoperiod. Fungal isolation on PDA medium frequency was 95% from diseases leaves. Morphological observations were made on 7- day- old single-spore cultures. The colonies initially appeared light grayish which turned sooty black in color. All fungal isolates were characterized by small, short-beaked, multicellular conidia. The conidia were ellipsoidal or ovoid and measured 9 to 25 µm × 5 to 10 µm (n = 40) with longitudinal and transverse septa. The morphological characters matched those of Alternaria alternata (Simmons et al. 2007). Genomic DNA of a representative isolate (FG01-FG03) was extracted using DNAzol reagent (Thermo Fisher Scientific MA, USA) and PCR amplification of the internal transcribed spacer (ITS) rDNA region, was performed with primers ITS1/ITS4 (White et al. 1990), partial RNA polymerase II largest subunit (RPB2) with RPB2-5F/RPB2-7cR (Liu et al. 1999) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene regions was performed with gpd1/gpd2 (Berbee et al. 1999). The obtained sequences were deposited in GenBank with accession numbers MW692903.1 to MW692905.1 for ITS-rDNA gene, MZ066731.1 to MZ066733.1 for RPB2 and MZ066728.1 to MZ066730.1 for GAPDH. BLASTn analysis showed 100% identity with the submitted sequences of A. alternata for ITS rDNA, RPB2, and GAPDH. To confirm pathogenicity, 2-month-old 15 healthy potted F. carica plants were sprayed at true leaf stage with conidial suspension by using an atomizer in a greenhouse. Each representative A. alternata isolate (FG01-FG03) was inoculated on every three plants with conidial suspensions (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) whereas, three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. All plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues and reisolation frequency was 100%. Re-isolated fungal cultures were again morphologically and molecularly identical to A. alternata, thus fulfilling Koch's postulates. Previously, A. alternata has been reported cause fruit disease of fig in Pakistan and California, USA (Alam et al. 2021; Latinovic et al. 2014). To our knowledge, this is the first report of A. alternata causing leaf spot on common fig in Pakistan. In Pakistan, fig is widely grown for drying, and this disease may represent a threat to fig cultivation.

Alternaria alternata causing Alternaria Leaf Spot of Cucumis melo (Muskmelon) in Pakistan.

In July 2019, leaf spot symptoms were observed on muskmelon (Cucumis melo L.) cv. Jackball-1 plants in an experimental field of 2.02 ha with a disease incidence of 30% (31°26'05.4"N 73°04'30.3"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms consisted of small, circular, brown, necrotic spots 1 to 2 mm in size covering 10 to 30% of the leaf blade, which gradually enlarged and developed concentric rings. To identify the causal agent of the disease, a total of 20 symptomatic leaves were collected. Small pieces removed from the margin between healthy and diseased tissues were surface disinfected in 70% ethanol for 2 min, rinsed three times with sterile distilled water, plated on Potato dextrose agar and incubated at 25 ± 2°C with a 12-h photoperiod. Morphological observations were made on 7-day-old single-spore cultures. The colonies initially appeared white and then turned olive-green. All 20 fungal isolates were characterized by small, short-beaked, multicellular conidia. The conidia were ellipsoidal or ovoid and measured 11.5 to 30 µm × 7.5 to 15 µm (n = 50) with longitudinal and transverse septa. Conidia were produced on short conidiophores in chains. The beaks were short (often less than one-third the body length) and conical or cylindrical. These morphological features concur with the description of Alternaria alternata (Fr.) Keissler (Woudenberg et al. 2013). For molecular identification, genomic DNA of four representative isolates (HMSMZA 07, 08, 09, 10) were extracted and PCR amplification of the internal transcribed spacer (ITS)-rDNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and translation elongation factor-1 alpha (TEF-1α) gene regions were performed (White et al. 1990, Berbee et al. 1999, Carbone & Kohn, 1999) respectively. The obtained sequences were deposited in GenBank with accession numbers MT253643.1-MT253646.1 (ITS-rDNA), MT318260.1-MT318263.1 (GAPDH), and MT318280.1-MT318283.1 (TEF-1α). BLASTn analysis of HMSMZA 07 sequences showed 100% identity with ITS rDNA (MN615420.1), GAPDH (MK637438.1) and TEF-1α (MN807795.1) sequences of A. alternata. To confirm pathogenicity, 5-6 weeks-old Muskmelon (Cucumis melo L.) cv. Jackball-1 plants (true leaf stage) were sprayed until runoff (1.5 to 2 ml per plant) with A. alternata conidial suspension (106 conidia/ml; obtained from 1 week-old cultures) amended with 0.1% (vol/vol) of Tween 20 using an atomizer in the green house. The experiment included four A. alternata isolates inoculated onto three muskmelon plants per each isolate, whereas control plants (n = 3) were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse and the experiment was conducted twice. After 5 to 7 days post inoculation, necrotic leaf spots were observed on the inoculated plants and A. alternata was reisolated and confirmed by morphological and molecular (ITS) features. No disease was observed on control plants. Previously, A. alternata on muskmelon has been reported in Pakistan (Ahmad et al. 1997), however this study provides a detailed description of disease symptoms, morphological and molecular identity of the causal agent including completion of Koch's postulates. The disease could represent a threat for muskmelon crop in Pakistan due to its increasing cultivation and therefore warrants the need to develop disease management strategies.

Occurrence of Leaf Spot Caused by Alternaria alternata on Eggplant (Solanum melongena) in Pakistan.

Eggplant (Solanum melongena L.) is a popular vegetable that is grown in both tropical and subtropical regions all year long. The crop is cultivated on small family farms and is a good source of income for resource-limited farmers in Pakistan. In early May 2019, leaf spots on eggplant (cv. Bemisaal) were observed in an experimental field (31°26'14.0"N 73°04'23.4"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, circular, brown, necrotic spots uniformly distributed on leaves. The spots gradually enlarged and coalesced into large, nearly circular or irregularly shaped spots that could be up to 3 cm in length. The center of the spots was light tan, surrounded by a dark brown ring, a chlorotic halo, and tended to split in the later developmental stages. Disease incidence was approximately 35% in the infected field. The causal agent of this disease was isolated consistently by plating surface sterilized (1% NaOCl) sections of symptomatic leaf tissue onto potato dextrose agar (PDA). After 6 days incubation at 25°C with a 12-h photoperiod, fungal colonies had round margins and the cottony mycelia were dark olivaceous with a mean diameter of 7.5 cm. For conidial production, the fungus was grown on potato carrot agar (PCA) and V8 agar media under a 16-h/8-h light/ dark photoperiod at 25°C. Conidiophores were septate, light to olive golden brown with a conidial scar, from which conidia were produced. Conidia were borne singly or in short chains and were obpyriform to obclavate, measured 29 ± 4.8 × 13.25 ± 2.78 µm (n=30) with zero to three longitudinal and two to six transversal septa. The morphological characters matched those of Alternaria alternata (Fr.) Keisel (Simmons et al. 2007). DNA was extracted using the DNAzol reagent (Thermo Fisher Scientific MA, USA). For molecular identification, internal transcribed spacer (ITS) region between ITS1 and ITS2, actin gene (ß-Actin), translation elongation factor (TEF-1α) gene, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of two representative isolates (JLUAF1 and JLUAF2) were amplified with primers ITS1/ITS4 (White et al. 1990), ß-Actin 512 F/783 R, EF1-728F/-986R (Carbone et al. 1999), and gpd1/gpd2 (Berbee et al. 1999), respectively. The sequences were deposited in GenBank (accession nos. MT228734.1 and MT228735.1 for ITS; MT260151.1 and MT260152.1 for ß-Actin, MT260163.1 and MT260164.1, for TEF-1a, and MT260157.1 and MT260158.1 for GAPDH). BLASTn analysis of these sequences showed 100% identity with the sequences of A. alternata for ITS rDNA, ß-Actin, TEF-1α, and GAPDH, respectively. Based on the morphological characters and DNA sequences, the leaf spot isolates of eggplant were identified as A. alternata. To confirm the pathogenicity on eggplant, six-week old healthy potted eggplants of cv. Bemisaal were sprayed at the true leaf stage with conidial suspensions of A. alternata (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) using an atomizer in the greenhouse. Three plants were inoculated with each of the two isolates (JLUAF1 and JLUAF2), whereas three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots which were similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling Koch's postulates. Previously, A. alternata has been reported to cause leaf spots on eggplant in India (Raina et al. 2018). To our knowledge, this is the first report of A. alternata causing leaf spot on eggplant in Pakistan. The disease could represent a threat for eggplant crops due to its increasing cultivation. It is important to develop disease management strategies for Alternaria alternata causing leaf spot of Eggplant in Pakistan.

First Report of Aspergillus niger causing Black rot of Grapes in Pakistan.

In June 2015 & 2016, a postharvest survey of table grapes (Vitis vinifera) cv. King's Ruby, was carried out in five different commercial fruit markets of Rawalpindi (33°38'19.2″N, 73°01'45.0″E) district, Punjab Province. Symptoms appeared as brownish lesions with black sporulation on grapes berries. The incidence of these symptoms on bunches ranged from 12 to 17% at all sites. Symptomatic tissue pieces were surface-sterilized with 0.1% sodium hypochlorite (NaOCl) for 30 seconds, rinsed three times with sterile distilled water, dried on filter paper for 45 seconds, and incubated on potato dextrose agar (PDA) at 25°C. After 3 days, dark brown to black mycelium were formed on PDA media. A total of 24 isolates were examined morphologically. The apex of the conidiophore was observed to be radiate. Vesicles were found to be spherical and covered with irregular metulae and phialides. Conidia were globose or subglobose measured (3.14 µm ± 2.24 in averaged diameter: n=50), dark brown to black, with roughened cell walls. The conidiophores were also smooth-walled, hyaline, and became melanized toward the vesicle. These characteristics of the fungus were similar to those described for Aspergillus niger van Tiegh (de Hoog et al. 2000). For molecular identification, the internal transcribed spacer (ITS) region, beta-tubulin (Bt) gene and partial RNA polymerase II largest subunit (RPB2) gene of representative isolate (Asp.n02) was amplified using primers ITS1/ITS4, BT2a/BT2b and RPB2-6F/RPB2-7R respectively (White et al., 1990; Glass & Donaldson, 1995; Liu et al. 1999). Sequences were deposited in GenBank (ITS, MN658871; Bt2, MT117924; and RPB2, MT318289). Based on BLAST analysis, sequences of the ITS region, Bt2 genes, and RPB2 gene showed 99 to 100% similarity of isolate Asp.n02 to Aspergillus niger (Accession Nos. MK307680.1, MN195121.1, MF078661.1 for ITS gene, MN567299.1, MK451029.1, MK451020.1 for Bt2 gene, and MK450788.1, MK450790.1 for RPB2 gene). To complete Koch's postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of isolate: Asp.n 02 was pipetted onto three non-wounded and four wounded (5 mm diam) asymptomatic grape berries cv. King's Ruby (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries similaries to serve as a negative control (Ghuffar et al. 2018; Jayawardena et al. 2018). Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice. Brownish lesions leading to black sporulation similar to the original symptoms were observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control. The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Aspergillus niger was reported previously in Europe and Israel causing mycotoxin (Ochratoxin A) OTA production on Table grapes (Bau et al. 2006). To our knowledge, this is the first report of Aspergillus niger causing black rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against the black rot of grapes in Pakistan.

First report of brown leaf spot of rice caused by Bipolaris zeicola in Pakistan.

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.

Conferring drought-tolerant wheat genotypes through morpho-physiological and chlorophyll indices at seedling stage.

The decrease in water resources due to the excessive use of water for irrigation purpose and climatic changes represents a serious world-wide threat to food security. In this regards, 50 wheat accessions were analyzed, using completely random factorial design at the seedlings stage under normal and drought stress conditions. Significant variation was detected among all accessions under both conditions. All characters studied showed variations in the mean values in water deficit environments in studied gemplasm at seedling stage. As seedling fresh weight, dry weight, relative water content, cell membrane thermo-stability, chlorophyll a & b were positively associated among themselves under drought conditions which showed the significance of these attribute for water deficit areas in future wheat breeding programs. Based on their performance, five accessions namely Aas-11, Chakwal-86, Pasban-90, Chakwal-97 and Kohistan-97 were selected as drought tolerant and three accessions namely Mairaj-08, Lasani-2008 and Gomal-2008 were selected as drought susceptible genotypes. The choice of wheat accessions based on the characteristics of the seedlings is informal, low-priced and less hassle. Likewise, the seedlings attributes exhibit moderate to high variation with an additive genetics effects on the environments. Best performance accessions under water deficit environment will be beneficial in future wheat breeding schemes and early screening for the attributes suggested in current experiment will be useful for producing best-yielded and drought-tolerance wheat genotypes to sustainable food security.

Assessment of grain yield indices in response to drought stress in wheat (Triticum aestivum L.).

Drought stress constricts crop production in the world. Increasing human population and predicted temperature increase owing to global warming will lead ruthless problems for agricultural production in near future. Hence, use of high yielding genotypes having drought tolerance and scrutinize of drought sensitive local cultivars for making them tolerant may be the proficient approaches to cope its detrimental outcomes. The current study was executed during 20015-2016 and 2016-2017 in field using randomized complete block design under factorial arrangements on 50 wheat genotypes for exploring their sensitivity and tolerance against drought. Some of the attributes of grain yield and drought tolerance indices were recorded. Grain yield showed negative correlations with tolerance index (TOL), drought index (DI) and stress susceptibility index (SSI) while positive correlation with mean productivity (MP) and geometric mean productivity (GMP) under drought condition. These findings depicted that tolerant genotypes could be chosen by high MP and GMP values and low SSI and TOL values. Based on the results, genotypes GA-02, Faisalabad-83, 9444, Sehar-06, Pirsabak-04 and Kohistan-97 were more tolerant and recognized as suitable for both normal and drought conditions. Genotypes of Chenab-00, Kohsar-95, Parwaz-94 and Kohenoor-83 confirmed more sensitive due to high grain yield loss under drought stress.

First record and taxonomic description of the genus Thysanoplusia (Fabricius) (Lepidoptera: Noctuidae: Plusiinae) from Pakistan.

Species belonging to genus, Thysanoplusia Fabricius of the subfamily Plusiinae which are polyphagous in nature and pest of vegetables, foods, legumes, fodder, fruits, ornamental plants and cotton crops. Samples were collected from different localities of district Bahawalpur. For collection, comprehensive and comparative surveys were carried out during 2017-18 on taxonomic account of species of the genus Thysanoplusia Fabricius and resulted identified one species Thysanoplusia orichalcea (Fabricius) first time from Pakistan. Morphological characters viz., vertex, frons, labial palpi, antennae, compound eyes, ocelli, proboscis, wing venation, male and female genital characteristics were used for the identification and classification. Dichotomous keys and photographs are also provided. There is hardly any substantial research work on taxonomic studies of subfamily Plusiinae Pakistan. So to fill this gap the present proposal was designed to study the diversity of Noctuid moths from Pakistan and very fruitful results have been obtained.