Collaborative Research on Shisham Die-back

Trees in Pakistan are dying from an unknown pathogen. Two UAF faculty researchers unite to discover the culprit and address the disease.

Dr. Siddra Ijaz (upper) and Dr. Imran ul-Haq (lower), from the University of Agriculture, Faisalabad.
Dr. Siddra Ijaz (upper) and Dr. Imran ul-Haq (lower), from the University of Agriculture, Faisalabad.

Dr. Siddra Ijaz (upper) and Dr. Imran ul-Haq (lower), from the University of Agriculture, Faisalabad.

Dr. Siddra Ijaz (upper) and Dr. Imran ul-Haq (lower), from the University of Agriculture, Faisalabad.

Scholar exchanges are the largest programmatic component of the U.S.-Pakistan Center for Advanced Studies in Agriculture and Food Security. With over 100 faculty members slated to travel from the University of Agriculture, Faisalabad (UAF), to the University of California-Davis, the program aims to develop UAF’s capacity to conduct cutting-edge research and train the next generation of Pakistani research scholars. For two members of the first exchange cohort, the experience led them to collaborate in a way that neither of them had previously considered.

Dr. Imran ul-Haq is a mycologist and Assistant Professor in the UAF Department of Plant Pathology. His research had previously focused on the etiology of perennial ornamental plants in Pakistan, but he wasn’t having much success using conventional plant pathology practices. In 2015, when the USPCAS-AFS announced the scholar exchange program, Dr. ul-Haq thought about how the opportunity might strengthen his research.

“Honestly, I was a conventional pathologist,” he said during a recent interview in his Faisalabad-based laboratory. “I didn’t know how to work with the molecular aspects. So when I submitted my proposal, I stated that I wanted to learn molecular research techniques at UC Davis.”

Meanwhile, Dr. Siddra Ijaz knew that she wanted to continue developing her skills in genotypic characterization. Dr. Ijaz is an Assistant Professor who specializes in genetic engineering at the Center of Agricultural Biochemistry and Biotechnology (CABB) at UAF. She received the first doctorate awarded from CABB in 2012, and in 2015 received a grant from the Pakistan Agricultural Research Board to develop a DNA index of mung bean genotypes. Dr. Ijaz was also nominated as a USPCAS-AFS exchange scholar, and she began working with Dr. Eduardo Blumwald to research drought-tolerance genes in Setaria viridis, or wild foxtail millet.

“There is no work reported on Setaria viridis regarding water stress or drought-resistant gene exploration,” stated Dr. Ijaz, sitting in her office at the CABB building on the UAF campus. “That was the very beginning of the first report on this plant’s gene profiles.”

While Dr. Ijaz was developing genetic expression profiles, Dr. ul-Haq was learning new techniques for preserving fungal samples from his exchange mentor, Dr. Lynn Epstein of UC Davis’s Department of Plant Pathology. Dr. Epstein had agreed to host Dr. ul-Haq during his exchange, and he was enthralled with the way she managed her lab space and fungal cultures. Her lab was using what is known as the dry filter paper technique, wherein fungal cultures are colonized on small pieces of filter paper and then slowly dried out over a month. Instead of six weeks of a viable culture, the samples could be stored for up to ten years. This was a revelation to Dr. ul-Haq.

“Nobody I knew was using that technique,” said Dr. ul-Haq. “What we had normally done was to isolate one fungal culture and grow it artificially on the petri plates, and we would revive it every six weeks. It was time-consuming, it was laborious, it took up all of our supplies, and the chances of contamination or specimen loss were high.”

While in Davis, Dr. ul-Haq and Dr. Ijaz began to talk to each other about the new methods they were learning and how academic research is approached in America. Dr. Ijaz laughed at the recollection. “During the exchange was our first interaction and first experience with each other,” she chuckled. “Our first meeting was at Davis. I’d never ever seen him at UAF before, and we’ve both been at UAF for seven or eight years. But as we talked, we came to learn that our research aligned, so we decided to write some proposals together.”

They approached Dr. Epstein about some research areas that they had brainstormed, and the three scientists agreed to submit applications the USPCAS-AFS collaborative research grants. One of the proposals to investigate the fungal origins of shisham die-back in Pakistan was approved and funded.

Shisham is a type of tree found throughout Pakistan, northern India, and Nepal. Originally planted to produce coal for steam engines, shisham is an important source of fuel and timber wood and is often placed along canal embankments to reduce erosion and increase soil fertility. In recent decades, many shisham trees have suffered from die-back, a disease that causes progressive death from the tips of twigs, branches, and roots. This has caused devastating impacts in forests and in countryside plantations as farmers reel from the economic impact of timber loss. While a species of Fusarium has been identified as the cause of shisham wilt, there is no consensus on what type of fungus may be causing die-back. The research project aims to lay the groundwork for a large-scale investigation into potential fungal culprits and types of genetic resistance in existing species.

“This project has two components,” states Dr. Ijaz, the principal investigator for the research. “The first component is preparing a survey of shisham germplasm and a survey of the different fungal isolates from affected trees. Dr. ul-Haq is doing the identification of the fungal pathogens from the affected plants. My role is based on cataloging the shisham germplasm and screening the genes that are involved in the infection cycle. Different expression profiling and different genomics approaches will be involved to explore what gene or what type of gene or what groups of genes are involved in resistance to shisham die-back.”

Dr. Siddra Ijaz stands in her office at the University of Agriculture, Faisalabad.
Dr. Siddra Ijaz stands in her office at the University of Agriculture, Faisalabad.

They both returned to Pakistan in the summer of 2016, each with a different plan of action. Dr. Ijaz began to collect samples of shisham trees around Pakistan, gathering over 60 plants from the province of Punjab and finding additional samples in Sindh, Baluchistan, and Khyber Pakhtunkhwa. “With shisham, nobody knows if it is one species or several species,” says Dr. Ijaz. “Nobody knows if there is one variety or many varieties. There is no data, no accession numbers, no variety numbers. So, after our survey, I added a diversity analysis so we can know for certainty whether it is one species or several.”

Dr. ul-Haq began preparing for the work of cataloging the suspect fungal culprits. “When I returned to UAF, I proposed establishing a fungal culture bank that would use the dried filter-paper technique that I learned on exchange,” he states. “I listed the problems that we were facing using our old preservation methods, and stated that we could work more efficiently with this new method. The proposal was approved and we were able to establish the fungal culture bank. We revised this fungal laboratory with new equipment and based the layout directly off of Dr. Epstein’s lab.” Dr. ul-Haq also converted an old storage closet next to his laboratory into a new cold storage space which could store freezers for fungal cultures, and purchased new molecular analysis equipment recommended by research scholars from UC Davis.

Scientist stands in lab room with freezers and cold storage systems
Once stacked high with desks and chairs, this converted storage room now holds Dr. ul-Haq’s cold storage equipment for his fungal culture bank at UAF.

They were now ready to proceed with the primary research objective. Dr. Ijaz elaborated on the next steps. “Now, in my laboratory, we are working on the micro-characterization of the diversity of the shisham plants that we collected from Punjab, and we are getting good results in our diversity analysis. After this, we will look at genetic resistance to the shisham die-back.

“Dr. ul-Haq is working to assess and analyze the fungal samples for potential causes. After he identifies the suspect fungus through pathogenic testing, he will send the fungal DNA to Dr. Epstein for DNA characterization. She will help confirm the fungal identity. Once Dr. Epstein identifies the exact fungus, Dr. ul-Haq will inoculate the selected shisham varieties which I am identifying. Once we artificially inoculate in greenhouse conditions, we hope to confirm the fungus as the cause of the shisham die-back. At that stage, my goal will be to screen the genes that are involved in the infection cycle, what genes are induced during infection, and hopefully identify what types of genes are involved in resistance to shisham die-back.”

Students sit at a lab bench surrounded by supplies
UAF graduate students work on preliminary research in Dr. ul-Haq’s fungal laboratory.

To help them with their laboratory analysis, the researchers convinced one of their own students to apply for a USPCAS-AFS exchange position so that he could help build the project’s research capacity. Hafiz Muhammad Usman Aslam, a doctoral student of Dr. ul-Haq’s, arrived at UC Davis in January of 2017 to learn more about molecular research techniques from Dr. Epstein and Dr. Pamela Ronald of the UC Davis Genome Center. “This is a prestigious opportunity to observe cutting-edge research,” said Mr. Aslam, who quantified fungal samples from Pakistan and began sequencing the results during his exchange. “I plan to help implement these techniques in my parent department at UAF.”

The research into the fungal cause of shisham die-back is still ongoing, but both professors cite their exchange visit to UC Davis as a truly transformational experience in their careers.

“I can’t express what Dr. Epstein means to me—she is still my mentor, even after my exchange,” says Dr. ul-Haq. “With her help, I was able to submit three USPCAS research proposals and we were awarded all three proposals. Now I am convinced that collaborative work is the most important thing if you want to have success. So, I have established collaborations with other colleagues and with other exchange research scholars.”

Dr. Ijaz elaborates: “There is a proverb in Urdu: ‘a frog in a well’. When you just have knowledge of your locality, or your thoughts are restricted, you just know about your boundaries. When you go out of these boundaries, you see different things, and you change your thinking. When I visited UC Davis, I found out there are more ways to think. The experience opened the doors for collaborations between colleagues. Without this exchange, these international collaborations would not exist.”

The U.S.-Pakistan Center for Advanced Studies in Agriculture and Food Security (USPCAS-AFS) works to enhance Pakistan’s economic growth and prosperity through applied research, academia-industry collaboration, and innovative academic programs.

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Profile written by Levi McGarry.