Impact of drought stress on simultaneously occurring pathogen infection in field-grown chickpea

Abstract

Drought stress and pathogen infection simultaneously occur in the field. In this study, the interaction of these two stresses with chickpea, their individual and combined effect and the net impact on plant growth and yield traits were systematically assessed under field and confined pot experiments. The field experiments were conducted for four consecutive years from 2014-15 to 2017-18 at different locations of India. Different irrigation regimes were maintained to impose mild to severe drought stress, and natural incidence of the pathogen was considered as pathogen stress. We observed an increased incidence of fungal diseases namely, dry root rot (DRR) caused by Rhizoctonia bataticola, black root rot (BRR) caused by Fusarium solani under severe drought stress compared to well-irrigated field condition. Similar to field experiments, pot experiments also showed severe disease symptoms of DRR and BRR in the presence of drought compared to pathogen only stress. Overall, the results from this study not only showed the impact of combined drought and DRR stress but also provided systematic data, first of its kind, for the use of researchers.

Figure 1

The morpho-physiological response of plants subjected to combined drought and F. solani infection. Chickpea genotype JG 62 was used to study the impact of combined drought and pathogen stress compared to drought only, and pathogen (F. solani) only stresses. Five days old nursery grown chickpea plants were used for drought, pathogen, combined stress treatments, and control. Chickpea root was immersed in F. solani spore suspension (1.1 × 10⁵ spores/ ml) for four hours to impose F. solani infection to pathogen only and combined stress treatments. For control and drought treatment, chickpea roots were dipped in sterile RO water for the same duration. Chickpea plants were re-planted into pots after four hours. Water withholding, to impose drought, was initiated five days after replanting into pots. Drought level (FC-35%) was achieved on 16th day after drought initiation and combined stress was counted from then. Disease symptoms at a morphological level were examined on fifth-day post combined stress treatment. Symptoms such as leaf yellowing (cyan arrow), root blackening, and root rot were observed in pathogen and combined stress treatment plants (A). Roots from control, drought, pathogen, and combined stress treatments were collected and observed under the 0.5 XPF objective of SZX16 Stereo Microscope (E). Scale bar represents 1 mm. Black root rot was evident in primary root (the distance between red arrows) of combined stress treatment. Additionally, pathogen treatment plants showed the emergence of lateral root (yellow arrow) and combined stress plants showed lack of lateral roots (white arrow). Leaf gas exchange parameters such as photosynthetic rate (B), stomatal conductance (C) and transpiration rate (D) were measured in the fourth leaf from the top in each treatment. Bar graph represents the average of three biological replicates ± SEM. Statistical significance was tested by two-way ANOVA followed by LSD All-Pairwise Comparisons post-hoc test. The different letters above each column represent significance difference between means at p < 0.05.

Figure 2

The morpho-physiological response of plants subjected to combined drought and R. bataticola infection. Drought, pathogen (R. bataticola) infection, and combined drought and pathogen infection were imposed on chickpea genotype JG 62. Chickpea plants for pathogen and combined stress treatments were grown in a sick pot containing R. bataticola inoculum. Plants for drought treatment and control were grown in autoclaved un-inoculated soilrite. Water with-holding for drought stress was initiated from fifth day post germination of chickpea. The desired drought (35% FC) level reached on the sixteenth day after water with-holding for both drought and combined stress treatments. Control and pathogen treatment plants were maintained at 90% FC for the entire experimental period. The drought was maintained at 35% FC for further five days, and disease symptoms at a morphological level were examined on the fifth-day. Symptoms such as leaf drying (cyan arrow), shedding of lateral roots, and root rot were observed in pathogen and combined stress-treated plants (A). Roots of control, drought, pathogen, and combined stress treatment plants were collected, and necrosis (black arrow), root rot (red arrow) and root diameter (blue arrow) was observed under the 0.5 XPF objective of SZX16 Stereo Microscope (E). Scale bar represents 1 mm. Leaf gas exchange parameters such as photosynthetic rate (B), stomatal conductance (C) and transpiration rate (D) were measured in forth leaf from the top for all the plants under four treatments. Bar graph represents the average of three biological replicates ± SEM. Statistical significance was tested by two-way ANOVA followed by LSD All-Pairwise Comparisons post-hoc test. The different letters above each column represent significance difference between means at p < 0.05.

Figure 3

Bright field microscopy root images showing high fungal colonization in xylem regions of plants under combined stress. Transverse hand sections of plant roots from individual and combined stress treatments were stained with lactophenol aniline blue and observed under 40X objective with fixed 40X condenser of LMI BM-X microscope. No fungal mycelia were observed in the xylem of control (A) and drought (B) treated plants. Plant roots treated with F. solani infection have less number of stained mycelia (C) whereas plants treated with combined drought and F. solani infection showed large number of stained mycelia (D). Plant roots with R. bataticola infection (pathogen only) showed less stained mycelia (E) whereas plants treated with combined drought and R. bataticola showed large number of stained mycelia (F). The cyan arrow indicates the aniline blue stained fungal mycelia. Scale bar represents 150 µm, microscopy was done with three technical replicates. Two independent experiments showed similar result. MX, metaxylem; PX, protoxylem; LXV, large xylem vessel; SXV, small xylem vessel; dotted line demarcates the metaxylem and protoxylem. Note: the blue arrows are for pointing out stained fungal mycelia and not for quantification. Further, number of stained mycelial structures were counted under each microscopic field and the values are 20 ± 6 (C), 94 ± 5 (D),19 ± 2 (E) and 53 ± 5 (F). Besides blue staining, the dark brown colour is due to infection (see Supplementary Fig. 23). The difference in diameter of xylem vessels between control and drought could be due to treatment effect. All the images are captured under white balance background.