IAA producing fungal endophyte Penicillium roqueforti Thom., enhances stress tolerance and nutrients uptake in wheat plants grown on heavy metal contaminated soils

Abstract

Heavy metals contaminated soil is a serious environmental concern that has a negative impact on agriculture and ecosystem. Economical and efficient ways are needed to address this problem worldwide. In this regard, exploration and application of proficient microbial strains that can help the crop plants to thrive in agricultural soils that are greatly contaminated with heavy metals. The present study mainly focused on the effect of IAA producing endophytic fungi Penicillium ruqueforti Thom., on wheat plants cultivated in soil rich in heavy metals (Ni, Cd, Cu, Zn, and Pb). P. ruqueforti has induced great resistance in wheat inoculated plants grown in heavy metal contaminated soil. Application of the isolated strain of P. ruqueforti restricted the transfer of heavy metals from soil to the plants by secreting indole acetic acid (IAA). Furthermore, P. ruqueforti inoculated wheat seedlings watered with waste water had higher plant growth, nutrient uptake and low concentrations of heavy metals in shoot and roots. On the contrary, non-inoculated wheat plants under heavy metal stress had stunted growth with symptoms of chlorosis. From the results, it is concluded that P. ruqueforti inoculation can establish a symbiotic relationship with host plants, which is useful for phytostabilization of heavy metals or in other words helping the host crops to flourish through soil that are highly contaminated with heavy metals.

Fig 1. Plant growth promoting activity of isolated fungal strain CGF-1 from halophytic plant S. surattense Burm.f.

+ive control = IAA mutant kernal (dek 18) maize line treated with distil water; -ive control = wild kernal (Dek 18) maize line grown under metal stress; CGF1 was applied to wild kernal (Dek 18) maize line grown under metal stress. Fig 1A represents shoot length; Fig 1B represents root length; Fig 1C represents chlorophyll content; Fig 1D represents weight of the plants; SL = shoot length; RL = root length; Chl = chlorophyll. The experiment was carried out in triplicate, each replicate comprised of 10 pots and each pot contained 6 seedlings (total = 6 × 10 × 3 = 180 seedlings per treatment). The boxplots having different letters are significantly different from each other at P < 0.05.

Fig 2. IAA estimation (μg/ml) in endophytic fungal strain CGF-1.

CGF-1 strain was grown on different sources and the produced IAA was quantified by using HPLC. Suc = sucrose; Yst = yeast; Glu = glucose; Malt = maltose; Trpa = 1000 μg tryptophan; Trpb = 500 μg tryptophan; Trpc = 100 μg tryptophan. The experiment was carried out in triplicate. The boxplots having different letters are significantly different from each other at P < 0.05.

Fig 3. Metal resistance assay of CGF-1.

Fig 3A = growth of CGF-1 on various concentration of nickle; Fig 3B = growth of CGF-1 on various concentration of cadmium; Fig 3C = growth of CGF-1 on various concentration of copper; Fig 3D = growth of CGF-1 on various concentration of Zn; Fig 3E = growth of CGF-1 on various concentration of lead; Ni = nickel; Cd = cadmium; Cu = copper; Zn = zinc; Pb = lead. The experiment was carried out in triplicate. The boxplots having different letters are significantly different from each other at P < 0.05.

Fig 4. NaCl resistance assay of CGF-1.

Fig 4A = fresh weight of CGF-1 grown on various concentration of NaCl; Fig 4B = dry weight of CGF-1 grown on various concentration of NaCl; FW = fresh weight; DW = dry weight. The experiment was carried out in triplicate. The boxplots having different letters are significantly different from each other at P < 0.05.

Fig 5. Biochemical contents of CGF-1 inoculated and non-inoculated wheat plant.

Fig 5A = proline, sugar and malondialdehyde contents of CGF-1 inoculated and non-inoculated wheat plant grown under heavy metal stress; Fig 5B = Chla, Chlb and carotenoid contents of CGF-1 inoculated and non-inoculated wheat plant grown under heavy metal stress; FIWP = fungal inoculated wheat plant; NIWP = non-inoculated wheat plant; Pro = proline; MDA = malondialdehyde; Chla = chlorophyll a; Chlb = chlorophyll b. The experiment was carried out in triplicate, each replicate comprised of 10 pots and each pot contained 6 seedlings (total = 6 × 10 × 3 = 180 seedlings per treatment). Means of fungal inoculated wheat plants (FIWP) were compared with non-inoculated wheat plants (NIWP) by using ttest; ‘***’ represents significant difference at P = 0.0005; ‘**’ represents significant difference at P = 0.005; ‘*’ represents significant difference at P = 0.05.

Fig 6. Antioxidant enzyme activities of CGF-1 inoculated and non-inoculated wheat plant.

FIWP = fungal inoculated wheat plant; NIWP = non-inoculated wheat plant; RG = reduced glutathione; Cat = catalase; PO = peroxidase; AA = ascorbate. The experiment was carried out in triplicate, each replicate comprised of 10 pots and each pot contained 6 seedlings (total = 6 × 10 × 3 = 180 seedlings per treatment). Means of fungal inoculated wheat plants (FIWP) were compared with non-inoculated wheat plants (NIWP) by using ttest; ‘**’ represents significant difference at P = 0.005; ‘ns’ represents non-significant difference at P = 0.05.

Fig 7. Heavy metals concentration of CGF-1 inoculated and non-inoculated wheat plants.

Fig 7A represents heavy metal concentration in shoots of CGF-1 inoculated and non-inoculated wheat plant grown in contaminated soil; Fig 7B represents heavy metal concentration in roots of CGF-1 inoculated and non-inoculated wheat plant grown in contaminated soil; FIWP = fungal inoculated wheat plant; NIWP = non-inoculated wheat plant; Ni = nickel; Cd = cadmium; Cu = copper; Zn = zinc; Pb = lead. The experiment was carried out in triplicate, each replicate comprised of 10 pots and each pot contained 6 seedlings (total = 6 × 10 × 3 = 180 seedlings per treatment). Means of fungal inoculated wheat plants (FIWP) were compared with non-inoculated wheat plants (NIWP); ‘***’ represents significant difference at P = 0.0005; ‘**’ represents significant difference at P = 0.005; ‘*’ represents significant difference at P = 0.05.

Fig 8. Nutrient uptake of CGF-1 inoculated and non-inoculated wheat plants.

CGF-1 inoculated and non-inoculated wheat plants were analysed for the nutrient uptake from the heavy metal contaminated soil; FIWP = fungal inoculated wheat plant; NIWP = non-inoculated wheat plant; Mg = magnesium; K = potassium; Na = sodium; Ca = calcium. The experiment was carried out in triplicate, each replicate comprised of 10 pots and each pot contained 6 seedlings (total = 6 × 10 × 3 = 180 seedlings per treatment). Means of fungal inoculated wheat plants (FIWP) were compared with non-inoculated wheat plants (NIWP) by using ttest; ‘**’ represents significant difference at P = 0.005; ‘*’ represents significant difference at P = 0.05.

Fig 9. Pollution load index and pollutant concentration factor.

Fig 9A represents pollution load index values of heavy metals in contaminated and reference soil; Fig 9B represents pollutant concentration factor values of heavy metals in fungal inoculated non-inoculated wheat plants in contaminated soil; FIWP = fungal inoculated wheat plant; NIWP = non-inoculated wheat plant; Ni = nickel; Cd = cadmium; Cu = copper; Zn = zinc; Pb = lead. The experiment was carried out in triplicate, each replicate comprised of 10 pots and each pot contained 6 seedlings (total = 6 × 10 × 3 = 180 seedlings per treatment). Means of fungal inoculated wheat plants (FIWP) were compared with non-inoculated wheat plants (NIWP) by using ttest; The experiment was carried out in triplicate, each replicate comprised of 10 pots and each pot contained 6 seedlings (total = 6 × 10 × 3 = 180 seedlings per treatment); ‘**’ represents significant difference at P = 0.005; ‘*’ represents significant difference at P = 0.05; ‘ns’ represents non-significant difference at P = 0.05.