Muhammad Ahsan

Onion is an important vegetable crop prone to many abiotic and biotic stresses. The blue mold rot of onion caused by Penicillium georgiense (P. georgiense) is one of the most damaging diseases in Pakistan and worldwide. The current study includes the factors that are responsible for losses at a considerable level where onion is being grown, transported, and stored. This study represents the first comprehensive integration of morphological and molecular identification of fungal pathogens isolated from onions (collected from different regions), showing the symptoms of blue mold rot. Standard tissue isolation and polymerase chain reaction (PCR) techniques were used for morphological and molecular characterization of the pathogen, respectively. Microscopic examination revealed the presence of short-chains of ellipsoidal to subspheroidal conidia, similar to the characteristic morphological features of P. georgiense. Further confirmation was done from sequencing of amplified Internal Transcribed Spacer (ITS) regions which showed 97–99% similarity to the P. georgiense species complex. Different biocontrol agents (BCAs) were evaluated against P. georgiense isolates under in vitro conditions, where Trichoderma sp., used as biocontrol agent, expressed maximum antagonistic effect. It significantly restricted the radial colony growth of the test fungus to only 37 mm, compared to control, where the colony reached upto 90 mm. In addition, other biocontrol agents such as Paecilomyces lilacinus (P.lilacinus) and Bacillus subtilis (B. subtilis) also demonstrated significant inhibitory effects on the radial colony growth of the test fungus. These BCAs inhibited the colonial growth to 59 and 67.67 mm, respectively. Based on the findings of the current study, P. georgiense is the significant causal agent of blue mold onion rot at all the studied locations, and can be efficiently controlled by using BCAs, a safe, environmental friendly and sustainable approach in agriculture.

Zucchini yellow mosaic virus (ZYMV) is a severe threat to cucurbit crops worldwide, including Pakistan. This study was pursued to evaluate the prevalence, geographic distribution, and molecular diversity of ZYMV isolates infecting cucurbits in Pakistan’s Pothwar region. Almost all the plant viruses act as a biotic stress on the host plants, which results in a yield loss. These viruses cause losses in single-infection or in mixed-infection cucurbit crops, and we have found a number of mixed-infected samples belonging to the Curubitaceae family. Serological detection of the tested potyviruses in the collected cucurbit samples revealed that ZYMV was the most prevalent virus, with a disease incidence (DI) at 35.2%, followed by Papaya ringspot virus (PRSV) with an incidence of 2.2%, and Watermelon mosaic virus (WMV) having an incidence as little as 0.5% in 2016. In the year 2017, a relatively higher disease incidence of 39.7%, 2.4%, and 0.3% for ZYMV, WMV, and PRSV, respectively, was recorded. ZYMV was the most prevalent virus with the highest incidence in Attock, Rawalpindi, and Islamabad, while PRSV was observed to be the highest in Islamabad and Jhelum. WMV infection was observed only in Rawalpindi and Chakwal. Newly detected Pakistani ZYMV isolates shared 95.8–97.0% nucleotide identities among themselves and 77.1–97.8% with other isolates retrieved from GenBank. Phylogenetic relationships obtained using different ZYMV isolates retrieved from GenBank and validated by in silico restriction analysis revealed that four Pakistani isolates clustered with other ZYMV isolates in group IIb with Chinese, Italian, Polish, and French isolates, while another isolate (MK848239) formed a separate minor clade within IIb. The isolate MK8482490, reported to infect bitter gourd in Pakistan, shared a minor clade with a Chinese isolate (KX884570). Recombination analysis revealed that the recently found ZYMV isolate (MK848239) is most likely a recombinant of Pakistani (MK848237) and Italian (MK956829) isolates, with a recombinant breakpoint between 266 and 814 nucleotide positions. Local isolate comparison and recombination detection may aid in the development of a breeding program that identifies resistant sources against recombinant isolates because the ZYMV is prevalent in a few cucurbit species grown in the surveyed areas and causes heavy losses and economic damage to the agricultural community.

Cucumber mosaic virus (CMV) is a tremendous threat to vegetables across the globe, including in Pakistan. The present work was conducted to investigate the genetic variability of CMV isolates infecting pea and spinach vegetables in the Pothwar region of Pakistan. Serological-based surveys during 2016-2017 revealed 31.70% overall CMV disease incidence from pea and spinach crops. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) revealed that all the positive isolates belong to CMV subgroup II. Two selected cDNA from ELISA-positive samples representing each pea and spinach crops were PCR-amplified (ca.1100 bp) and sequenced corresponding to the CMV CP gene which shared 93.7% nucleotide identity with each other. Both the sequences of CMV pea (AAHAP) and spinach (AARS) isolates from Pakistan were submitted to GenBank as accession nos. MH119071 and MH119073, respectively. BLAST analysis revealed 93.4% sequence identity of AAHAP isolate with SpK (KC763473) from Iran while AARS isolate shared maximum identity (94.5%) with the strain 241 (AJ585519) from Australia and clustered with some reference isolates of CMV subgroup II from UK (Z12818) and USA (AF127976) in a Neighbour-joining phylogenetic reconstruction. A total of 59 polymorphic (segregating) sites (S) with nucleotide diversity (π) of 0.06218 was evident while no INDEL event was observed in Pakistani isolates. The evolutionary distance of Pakistani CMV isolates was recorded as 0.0657 with each other and 0.0574-0.2964 with other CMV isolates reported elsewhere in the world. A frequent gene flow (Fst = 0.30478 <0.33) was observed between Pakistani and earlier reported CMV isolates. In genetic differentiation analysis, the value of three permutation-based statistical tests viz; Z (84.3011), Snn (0.82456), and Ks* (4.04042) were non-significant. The statistical analysis revealed the values 2.02535, 0.01468, and 0.71862 of Tajima's D, Fu, & Li’s F* and D* respectively, demonstrating that the CMV population is under balancing selection.

Garlic (Allium sativum L.), an aromatic bulb crop, has long been recognized all over the world as a valuable vegetable for foods and a popular remedy for various ailments and physiological disorders. In Pakistan, garlic is one of the most important members of alliums vegetables and ranked at second position after onion. The average garlic crop yield in Pakistan is often low and variable due to many viruses (Gilani et al. 2016). Viral diseases of garlic are worldwide in nature which can reduce the yield up to 50% during successive cultivation (Chodorska et al. 2014). Like other Allium species, garlic can be host of Iris yellow spot orthotospovirus (IYSV, genus Orthotospovirus, family Tospoviridae), and infections are common throughout the world (Karavina et al. 2016; Bag et al. 2015). In March 2016, about a quarter of all garlic plants in farmer’s fields from Capital Territory Islamabad, Pakistan, showed symptoms of pale yellow or straw colored, spindle-shaped spots leading to collapsed brown larger patche...