Water Sharing Conflicts and Management in the Indus River Basin

Sharing water resources within country and amongst transborder countries often create conflict because of increasing demand of fresh water for their domestic, industrial and agricultural sectors due to growing population and increasing economic activities. As a result, every country is interested to build more water storages like dams and barrages to safeguard their water requirements in the lean periods or to protect their areas during flood period. Therefore, a transboundary conflict amongst riparian countries on water sharing is obvious facts which are resolved either through bilateral dialogue or by involving international arbitrators. Similarly, a conflict of water sharing within a country has also been serious issue particularly during drought and lean period resulting political conflicts and obstacles in construction of dams and reservoirs. Pakistan is country of 207 million populations, the sixth of the most populated country of the world has been facing transboundary water sharing conflict with India while within a country inter provinces mistrust over water distribution has created reservation over the construction of new water storages. Pakistan has two agreements which provide legal framework for water distribution and management. Indus Water Treaty is an international agreement signed in 1960 between India and Pakistan and other is national agreement amongst the provinces called Indus water accord signed in 1991 by province. Despite several reservations and hostile territorial conflicts between India and Pakistan the Indus water treaty has been successfully functioning in managing water distribution of Indus River and its eastern tributaries originate from Indian occupied Kashmir. Similarly, Indus water accord 1991 provides a mechanism to resolve water sharing conflicts amongst provinces.

Study of Aflatoxigenicity and Biocontrol of Aspergillus Flavus and Impact of Imizoquins on its Physiology

Aspergillus flavus is a world-wide threat to human, animal and plant health due to the production of aflatoxins in many food and feed products. In first phase of the study, the incidence of aflatoxigenic strains of A. flavus was evaluated in cattle feed, water and milk from three districts of Punjab province of Pakistan (Rawalpindi, Gujrat and Okara) due to high incidence of aflatoxins in dairy products of those areas. In total, 39 of the hundred samples collected were contaminated by A. flavus. Specifically, the incidence of A. flavus was 54.28 %, 53.3 % and 11.42 % in feed (n=35), water (n=30) and milk (n=35) samples, respectively. These strains were further investigated for their aflatoxigenic nature using cultural (fluorescence under UV-light and NH4OH vapor induced color change test) and molecular (PCR) methods. The UV method indicated aflatoxigenic potential in 62 % of strains, and the ammonia vapor test showed 54 % of samples to be positive for aflatoxin production. The UV test is more sensitive than the ammonia test; however, comparable results from both methods strengthened our confidence in the findings. PCR detection of aflatoxin producing cluster of A. flavus was done by employing primers for four structural genes i.e. nor-1, ver-1, omt-A, aflR and two primers were used to distinguish A. flavus from A. parasiticus. The omt-A and aflR genes were regarded as potential markers for aflatoxins production because these genes were amplified in all those strains that were regarded as aflatoxigenic based on cultural methods for aflatoxin detection. It was demonstrated that, in addition to feed, water also acts as a potential threat for aflatoxigenic A. flavus entry into the food chain of the studied region. Moreover, the combined use of cultural and molecular methods, used in this study, can provide a cheaper and faster way to detect aflatoxigenic A. flavus in food and feed samples in developing countries. Fungal secondary metabolites have long been investigated for their pathogenic and therapeutic roles. Numerous biosynthetic gene clusters (BGCs) have been studied in order to decipher the roles of these genetic clusters. A previous study found that Ralstonia solanacearum, a soil-borne bacterium produces a lipopeptide, ralsolamycin, that lowers the expression of a BCG, named as imq, in A. flavus. The transcription factor of this gene cluster, imqK, regulates the synthesis of tripeptide-derived alkaloids, imizoquins. In second phase of this study, imizoquins were found to promote spore germination in A. flavus and few related fungi by acting as endogenous antioxidants to provide protection against germination inhibitory effects of reactive oxygen species (ROS). In addition to their role in fungal development, imizoquins also counteract the delaying effects of ralsolamycin extracts on germination and inhibit the growth of R. solanacearum in a bacteriostatic manner. Thus, this study highlighted the role of secondary metabolites in bacterial-fungal interactions. In third phase of the study, the use of generally regarded as safe (GRAS) microorganisms for the biocontrol strategy against aflatoxigenic A. flavus was investigated. Thirteen Geotrichum candidum strains were used in the study, of which ten strains were locally isolated from dairy products and three strains were procured from University of the Caen, France. These strains were individually assessed for anti-A. flavus activity as well as in combination with four lactic acid bacteria (LAB) strains which included three Lactococcus garvieae strains QAULG01, QAULG02, QAULG03 and a Lactococcus lactis strain QAULG04. The antagonistic potential of G. candidum strains and their combinations with the lactic acid bacteria were assessed by well diffusion assays. G. candidum strain (QAUGC01) and the combination of QAUGC01+QAULG01 gave the best antagonism and yielded the inhibition zones of 19 mm and 30 mm, respectively. The organic extracts obtained from the cell free supernatants of the two samples i.e. QAUGC01 and QAUGC01+QAULG01 yielded the most promising results, and the organic extract from QAUGC01 provided 20 mm inhibitory zone against A. flavus mycelial growth. One of the active metabolites of the cell free supernatants of the two samples was identified to be a derivative of butanoic acid. In conclusion, it is demonstrated through these studies that microbial interaction studies provided useful information regarding the small biomolecules that microorganisms (e.g. LAB from cattle gut, G. candidum from dairy products and R. solanacearum from rhizosphere) use to antagonize and halt the proliferation of nearby residing microbial competitor (e.g. A. flavus in this study).