by Chad Redman
Genetically modified organisms have already revolutionized the global food production industry. However, it is important to continue searching for new opportunities to develop further genetically modified crops and livestock. In that spirit, we will examine this publication by Nagaraju et al. (2012), who test the interaction between three different strains of Trichoderma harzianum bacteria and the seeds of sunflowers. Using a disease susceptible sunflower variety, the researchers coated seeds with these strains of bacteria and observed its effects on disease rate, growth rate, nutrient uptake, and seed production of grown plants. Overall, coating seeds with Trichoderma harzianum proved to significantly benefit the plants, although there was also substantial variation between the different bacterial strains. This finding is highly interesting in the search for more developed genetically modified crops because it introduces many questions. What is it about the bacteria that lead to enhanced plant performance? What genes are responsible? What are the products of those genes? Ultimately, can we isolate and introduce those genes into the plants themselves? This is the beginning of a long line of research to come.
Nagaraju et al. investigated the effects of coating sunflower seeds in three different isolates, or strains, of Trichoderma harzianum bacteria, using several metrics that included resistance to sunflower downy mildew disease, growth rate, seed production and more. The fundamental procedure included growing both treated and non-treated (control) plants in greenhouse and field conditions.
Nagaraju et al. began with the isolation of Morden sunflower seeds, a highly disease vulnerable variety of sunflower, as well as the pathogen Plasmopara halstedii, which is the cause of sunflower downy mildew disease. The seeds were procured from a seed company in India, and were then grown under greenhouse conditions in soil that contained P. halstedii. Researchers collected tissues from these plants as they grew, from which they were able to isolate spores of P. halstedii.
Next, Nagaraju et al. separated T. harzianum from the soil of an active farm in India and cultured the bacteria under laboratory conditions. From this original sample, three distinct strains of T. harzianum were identified and cultured. They were: PGPFYCM-2, PGPFYCM-8, and PGPFYCM-14. Importantly, the researchers performed polymerase chain reaction (PCR) to strictly define the genetic makeup and variance of these three bacteria.
Armed with the proper seed and the necessary bacteria, Nagaraju et al. began to treat the seeds. This step of their procedure was fairly complex, but may be understood as coating Morden sunflower seeds with three different concentrations of T. harzianum. The first concentration was produced by submerging seeds in a solution that contained spores of T. harzianum and allowing them to dry with the spores still on the seed. Specifically, this solution contained 1×108 spores per mL. The other two concentrations involved powdering seeds in a layer of talc, a solid that can be made into a very fine dust. The talc powder contained 2.1×107spores per gram of talc, while eight and ten grams of this powder respectively were applied to one kilogram ofseed, representing the next two concentrations of T. harzianum treatment. All three strains of T. harzianum were used to treat seeds with each of these concentrations separately.
At this point, the actual experiments could begin. PGPFYCM-2, PGPFYCM-8, and PGPFYCM-14 coated seeds at all three concentrations were grown in wet paper towels under laboratory conditions to measure root growth, shoot growth, and germination as a percentage of seeds. Growth was given seven days, and the vigor of the seed germination was calculated for each treatment.
Next in line was the disease resistance experiment. Under greenhouse conditions, and in sterile soil, seeds of Morden coated in all three concentrations of the three strains of T. harzianum were planted in pots, being separated based on concentration and bacterial strain. After reaching seedling level of maturity, the plants were exposed to P. halstedii and the percentage of each treatment that displayed symptoms of sunflower downy mildew disease were recorded.
Additionally, using the same experimental design consisting of individual pots containing sterile soil for each of the bacterial strain treatments at each concentration, Nagaraju et al.investigated how well plants used nutrients in the soil. Nitrogen, phosphorus and potassium were the three macronutrients tested for in separate experiments. After 30 days of growth under greenhouse conditions, plants from each experimental group were harvested and subjected to testing for the concentration of N, P or K present in the plants’ cells.
Finally, the authors conducted field experiments on an active farm, although under controlled water and nutrient conditions, to test the growth traits of sunflowers treated with P. halstedii. Again, PGPFYCM-2, PGPFYCM-8, and PGPFYCM-14 coated seeds at all three concentrations were grown separately over the full course of the sunflower life cycle. At 30 days, researchers recorded the incidence of downy mildew disease. Then, time till first flowering and plant height, crop duration, and earhead diameter at maturity were recorded, and seed weight was measured after harvest for each experimental group.
Results from this study were complicated by the use of nine variables, including three different strains of T. harzianumbacteria and also three different concentrations of the bacteria. However, results from each of the experiments showed a positive relationship between seed treatment with T. harzianum and plant growth and survival.
Specifically, in the initial germination vigor test, there were significantly different rates of germination between all treatments, with PGPFYCM-14 showing the greatest germination at 91%, followed by PGPFYCM-2 (90.25%), PGPFYCM-8 (88.5%), and last the control. Also, the concentrations displayed significant differences, with solution-treated seeds germinating most often, followed by the 10 grams talc per kilogram seed, and then the 8 grams talc per kilogram seed concentration.
As for degree of disease resistance, treated seeds far outperformed the control. Each treatment also differed significantly, with PGPFYCM-14 showing the greatest resilience, followed by PGPFYCM-2 then PGPFYCM-8. Similarly, the different concentrations of T. harzianum were correlated with different levels of protection. Solution-treated seeds were the best off, and the 8 grams talc per kilogram seed bested the 10 grams talc per kilogram seed treatment.
In terms of nutrient uptake, the results are split up between nitrogen, phosphorus and potassium. For nitrogen uptake, there was no significant difference between the three types of T. harzianum bacteria, however, the concentrations did differ significantly. While the control group was found to have the lowest amount of nitrogen, solution treated seeds boasted the greatest nitrogen content, followed by the 8 grams talc per kilogram seed and then the 10 grams talc per kilogram seed treatment.
Phosphorus uptake did vary significantly based on the treatment of T. harzianum. PGPFYCM-14 had the greatest uptake of phosphorus, then PGPFYCM-2 and lastly PGPFYCM-8. Concentration of treatment had a significant impact here as well, revealing that solution treated seeds once again outperformed the others, with 8 grams talc per kilogram seed treatment being least effective. Interestingly, identical results were established for potassium uptake.
On to field testing, the different strains of T. harzianum yielded different levels of disease resistance just as they did in greenhouse testing. PGPFYCM-14 treated seed demonstrated the greatest resistance, followed by PGPFYCM-2, and PGPFYCM-8 treated subjects proved to have very little resistance to disease beyond what the control group exhibited. Likewise, the three concentrations of treatment produced significantly different results, with solution-treated seeds outdoing the rest and 8 grams talc per kilogram seed contributing the lowest resistance to disease.
Furthermore, plant growth in the field varied significantly dependent on metric, strain, and concentration. As for height of the plant, PGPFYCM-14 plants grew the tallest, then PGPFYCM-2, PGPFYCM-8, and finally control plants. In terms of concentration, the solution-treated seeds grew tallest followed by 8 grams talc per kilogram seed and then the 10 grams talc per kilogram seed. Time to crop maturity followed the same pattern for performance based on T. harzianum. However, by this metric, the 10 grams talc per kilogram seed outperformed 8 grams talc per kilogram seed, with solution-treated seed once again proving most effective.
For time to first flowering, earhead diameter, and seed mass measurements, PGPFYCM-14 treated plants were the earliest, largest and heaviest, followed by PGPFYCM-2 and finally PGPFYCM-8. However, they did not follow identical patterns for response to different treatment concentrations.
The earliest flowers were produced by solution treatment, just before 10 grams talc per kilogram seed and with 8 grams talc per kilogram seed producing flowers the slowest. In contrast, the heaviest seeds formed on plants treated with 8 grams talc per kilogram seed, whereas the lightest seeds came from solution-treated plants. And finally, earhead diameter of the sunflowers decreased from the 10 grams talc per kilogram seed treatment to the solution treatment, and with the smallest earheads on 8 grams talc per kilogram seed treated plants.
Nagaraju et al. conducted an extremely lengthy, multifaceted procedure to clarify the effect of a beneficial bacteria coating on sunflower seeds. Truly, they have proven that coating these seeds will result in many benefits. However, the researchers have done more than that. They have opened the door for research on how to accomplish the same task artificially. How can we engineer seeds to inherently possess the desirable traits demonstrated in research by Nagaraju et al? This sort of example is exactly why genetically modified organisms were first imagined, and the origin of future genetic advancement of crops and livestock.
Nagaraju, A., Sudisha, J., Murthy, M. S., Ito, S. 2012. Seed Priming withTrichoderma harzianum Isolates enhances Plant Growth and Induces Resistance Against Plasmopara halstedii, an Incitant of Sunflower Downy Mildew Disease. Australasian Plant Pathology 41, 609-620.