A rhizobacterium found in the Thar desert in India has the potential to become an environmentally friendly biofertilizer while also protecting plants against drought stress.
The bacterium, Peribacillus frigoritolerans T7-IITJ, which was isolated from the rhizosphere of the wild indigo plant Tephrosia purpurea, was found during the search for a stress-tolerant microbe that could promote the growth of arid region crops under stressed conditions.
The investigation into the capabilities of the bacterium has resulted in a study, ‘Peribacillus frigoritolerans T7-IITJ, a potential biofertilizer, induces plant growth-promoting genes of Arabidopsis thaliana’, which was published in the Journal of Applied Microbiology, an Applied Microbiology International publication.
Arid region
“In 2023, my PhD student Debankona started to work on the rhizobacteria of the Thar desert in search of a stress-tolerant microbe which will promote the growth of arid region crops under stressed conditions and therefore can be used as a biofertilizer in the near future,” said corresponding author Dr Ayan Sadhukhan, assistant professor at the Indian Institute of Technology Jodhpur.
“We successfully isolated Peribacillus frigoritolerans from the rhizosphere of Tephrosia purpurea which enhanced Arabidopsis growth under non-stressful and drought stress conditions through the upregulation of root growth, defense including jasmonate signaling, and nutrient mobilization-related genes but downregulation of well-known drought-responsive genes like those involved in ABA and ethylene signaling pathways, and yet provided protection against drought stress. Relatives of this bacterium had been found earlier in the Sahara desert of Africa, as well as in the permafrost of Antarctica.”
Biofertilizer trend
Almost 33% of the world is desert and due to an ever-expanding population in the face of global warming and climate change, it is necessary to come up with a rational solution to the problem, Dr Sadhukhan said.
“Nowadays people are more inclined towards the use of biofertilizers rather than chemical ones. Our motto is to promote sustainable agriculture and our plan was to isolate some plant growth promoting stress tolerant rhizobacteria from the rhizosphere of the Thar desert plants which can be applied as a biofertilizer in the arid region plants.
“It will be cost effective and will not cause any hazard to the environment; in addition to that it will also promote the growth and stress tolerance of our desired crops. We also tried to find out the mechanism of action of the rhizobacteria by means of which they promote plant growth under drought stress.”
Bacteria from rhizosphere
The team worked to isolate plant growth promoting rhizobacteria (PGPR) from the rhizosphere of the native desert plants by collecting rhizosphere soil samples, diluted them in phosphate buffer saline and spreading them in LB agar.
“After repeated streaking from single colonies we successfully isolated three pure cultures. Their stress tolerance was estimated by growing them in LB broth supplemented with polyethylene glycol 6000, in nutrient deficient condition and in both high and low temperatures and extremes of pH,” Dr Sadhukhan said.
“We analysed the biochemical characteristics of our isolated microbes by catalase, MR-VP and starch hydrolysis tests. We also tested their ability to solubilize phosphate and produce IAA and EPS. Then for identification of the isolates we performed rDNA sequencing and two of them were found to be human pathogens. Hence, we had to proceed with Peribacillus frigoritolerans T7-IITJ.
“For further understanding of the genes present in our isolate, we performed whole genome sequencing. We also analysed the nitrate and siderophore producing ability and antifungal activity of T7-IITJ.
Hydroponic application
“We assessed the plant growth promoting ability of our isolate by applying it in Arabidopsis thaliana hydroponically. We also estimated the chlorophyll, proline and reactive oxygen species (ROS) content of Arabidopsis leaves on application of our isolate under drought stress. Finally, we performed a transcriptome analysis on 14-day-old drought-stressed Arabidopsis inoculated with T7-IITJ to understand the mechanism of action of the microbe.”
The key findings were as follows. T7-IITJ, a gram +ve bacillus species, was found to be highly tolerant to drought, nutrient deficiency, high temperature and pH. It can solubilize phosphate, nitrate and to produce siderophores which was confirmed by the presence of those genes in the genome of T7-IITJ.
It also showed biocontrol activity against two fungal pathogens of plants Rhizoctonia solani and Fusarium oxysporum. It was able to produce IAA-like compounds and exopolysaccharides which points towards the biofilm formation of the microbe around the plant roots and increased water permeability.
Colonization of roots
“FE-SEM imaging of Arabidopsis roots showed the colonization of T7-IITJ on the surface of the roots of the plant and we also found the internal root tissue colonization in Tephrosia purpurea, the host plant,” Dr Sadhukhan said.
”It significantly improved the growth of Arabidopsis under drought stress as well as in controlled environment. On inoculation of T7-IITJ under drought stress the chlorophyll and proline content of Arabidopsis increased but ROS production decreased indicating its protective role.
“The transcriptome analysis revealed the upregulation of root length, root hair, nutrient mobilization, and defense-related genes. On inoculation of our isolate germination of wheat (Triticum aestivum) and foxtail millet (Setaria italica) was found to be significantly improved.”
Surprising finds
But there were some surprises. Transcriptome analysis revealed the downregulation of drought responsive genes like those in ABA, ROS, senescence-related pathways etc. yet an elevated drought stress response.
“Reduced expression of these genes indicates that the endophyte colonizes in the root of the plants systematically thereby not overexpressing those genes but still elevating the stress tolerance. It also helps in the reduction of the drought stress phenotype,” Dr Sadhukhan said.
“The drastic phenotype of Peribacillus frigoritolerans-inoculated Arabidopsis under drought stress compared to the only drought stressed plants was the main motivation behind this study and the above-mentioned thing surprised us.”
Green revolution
Describing PGPR as a ‘fresh green revolution” worldwide, Dr Sadhukhan said this was one of many studies conducted on different PGPR around the world with much yet to be discovered.
“Global food security and climate change is becoming a big issue in today’s world, hence, to meet the needs of the growing population, we must come up with a sustainable solution which can be a biofertilizer.
“Moreover, microbes from arid regions are more tolerant to different abiotic stresses so desert areas are a hub of stress-tolerant bacteria. We wanted to exploit the benefit of staying near the Thar desert and the diverse microbial population which is native to the Thar desert to discover unknown facts and to produce a biofertilizer which will not only be useful to us but to the agriculture of the arid regions worldwide.”
Fulfilling its potential
If Peribacillus frigoritolerans is to fulfil its potential as a PGPR for developing biofertilizer, the isolate must be tested in field conditions to analyse its efficacy, Dr Sadhukhan said.
“It might also be used with other isolates, provided their coexistence is compatible, to be applied as a consortium in the field. It should be tested in arid region crops - as we have seen it improved the germination rate of some crop species.
“After all this testing it can be commercialised. In addition to that, knockout studies can be conducted on the microbe to discover the genetic regulation of downregulated and upregulated genes. Why were drought responsive genes downregulated, yet it still provided protection against drought - that question is still unanswered. We found a possible explanation in very few papers, but it is yet to be validated.”
Funding from IIT Jodhpur (I/SEED/ASK/20220015), SERB, Govt. of India (SRG/2022/000169), and Office of Principal Scientific Advisor to the Govt. of India (JCKIF/Thar/Proj-01/2022) are gratefully acknowledged.
The article, ’Peribacillus frigoritolerans T7-IITJ, a potential biofertilizer, induces plant growth-promoting genes of Arabidopsis thaliana’, appears in the Journal of Applied Microbiology.
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