Impact of fungi and bacterial-based bio-fertilizers on biochemiacal and molecular changes and induced resistance of checkpea under fusarium wilting disease

Chickpea is an important grain legume in Asia, and makes a significant contribution to the food and nutrition security of the people. Fusarium wilt is an important disease of chickpea, causing significant yield loss. This malady is the most important and destructive soil-borne disease of chickpea in Iran. Excessive use of chemical fungicides to control this soil-born pathogen can have adverse effects on human health and environment, and can lead to fungal resistance to fungicides. Increasing induced resistance using resistance inducers as bio-fertilizers can be considered as an alternative method to plant disease control. The use of nitroxine a bio-fertilizer containing Azosprillium and Azotobacter species as well as mycorrhizal fungi to induce systemic resistance mechanisms has been demonstrated in different plants under biotic stress. The aim of this study was to investigate the effect of bio-fertilizers to induce acquired resistance in chickpea plant under pathogen stress as compared to non-infected plants.

The change of some antioxidant enzymes and gene expression analysis was examined to evaluate the effect of biofertilizers on increasing resistance in chickpea plants infected with Fusarium oxysporum fsp. ciceri. A factorial experiment based on complete randomized block design with three replications was conducted under greenhouse conditions in 2014. The eight treatments included: 1) Plant control without infection (sh), 2) Fusarium oxysporum (Foc), 3) Mycorrhiza (Gi), 4) Nitroxin (N), 5) Mycorrhiza+Nitroxin+Infection (GI+N+F), 6) Mycorrhiza+Nitroxin (GI+N), 6) Mycorrhiza + Fusarium oxysporum (Gi+F) and 8) Nitroxin+Fusarium (N+F). Mycorrhiza, Fusarium oxysporum and Nitroxin+Fusarium (M+F+N) were used in this study. Catalase, Proxidase, and Polyphenoloxidase activity was assessed by spectrophotometer with corresponding wavelengths. Total RNA was isolated using an extraction kit, according to the manufacturer’s protocol. Real-time RT-PCR was performed in a thermocycler using the following program: 5 min at 94 °C, followed by 35 cycles of 30sec at 95°C, 30 min at 59°C and 30 min at 72 °C, with final extension for 10min at 72°C. All acquired data were analyzed using SAS software version 9 and mean values were compared using Duncan's multiple range test. The changes of transcript level were measured via a comparative technique. Actin genes were used as internal reference.

Data analysis revealed that the highest rate of total phenol and protein (50/292 mg/mL) in treated plants was related to nitroxin and mycorrhiza treatments, respectively. The greatest change in all three enzymes was observed in mycorrhiza treatment 72h after plant infection. Mean comparison of gene expression data at different time intervals showed a high expression level 72h after plant infection in mycorrhizal application. Our results indicated that the maximum effect of bio-fertilizer application occurred 72h following plant infection with the pathogen. Application of both biofertilizers without Fusarium infection did not show any significant change in the expression level of the two tested gene.

Biofertilizer agents can improve plant growth through several different mechanisms such as protecting the plant under stressful conditions and defense against plant pathogen which can lead to reduced disease and death. The increase of total phenol and protein as the precursor of plant defense mechanism through apply biofertilizers in infected plants indicated the efficiency of these compounds to reduce disease. Antioxidant enzymes as a biomarker were used to evaluate the effect of exogenic compounds on increasing plant resistance against biotic stress. As with a similar work, we found a correlation between biofertilizer application and enzyme activity. Other parameters such as phenotypic characters and yield components can be used as a marker to investigate the effect of biofertilizers in these domains. Analysis of variance of the data related to gene expression confirmed the efficiency of biofertilizers on inducing resistance. Based on our results, we recommended use of biofertilizers in a single or mixture form to improve growth conditions in stressed plants especially under biotic stress. To make the bio-fertilizer more efficient in the field, a coordinated work by different science domains as bacteriology, chemistry, genetics, and agronomy as well as farmers as bio-fertilizer consumers seems necessary. This coordination could facilitate the adaptation of fungi and bacterium-based bio-fertilizers to different agriculture systems.