In the present study, bioactive endophytic fungal strains were initially screened for their growth potential in Cd and Cu polluted mediums. The results suggested that P. funiculosum had higher growth rate as compared to the other three M. anisopliae, Promicromonospora sp. and Exophiala sp endophytes. P. funiculosum had higher potential to dwell in excessive Cu than Cd. The role of endophytic fungi has been recently elucidate by Li et al.  however, this more concentrate on the bacterial strains than fungal strains. Few endophytes like Microsphaeropsis, Mucor, Phoma, Alternaria, Peyronellaea, Steganosporium, and Aspergillus have been known to grow well in polluted medium and protect plants from adverse effects of metal stress . Very little has been known about endophytic Penicillium and its role in host-plant resistant to metal stress. Some strains of Penicillium janthinellum and P. simplicissimum have been found to grow well in high Cu medium , suggesting that they bears higher biosorption capacity against Cu. Previous studies showed that some strains of Penicillium can extend tolerance to host plants against metal toxicity. For example, strains of P. janthinellum and P. simplicissimum reduced the aluminum and zinc toxicity as it produced citric acid . Similarly, an isolate of Penicillium sp. bioaccumulated Cd during incubation period . The findings of studies suggest that strains/species of Penicillium can mitigate the Cd and other metal-related toxicity, which can be attributed to their potential to produce bioactive metabolites or enzymes [30, 31]. Previously, it was noted that P. funiculosum produce bioactive gibberellins which can contribute to the ability of a fungus to convert the toxic metal into stable complexes . A similar effect of stabilizing the negative effects of sodium chloride toxicity was also observed when this endophyte was inoculated to soybean plants .
Association of endophytic fungi with crop plant can increase plant fitness under abiotic stress conditions [11, 13, 32]. The secretion of plant growth regulating compounds by the endophyte probably is the mechanism responsible for the enhancement of plant growth [11, 13]. Since the culture filtrate of P. funiculosum showed the presence of physiologically active gibberellins , therefore, we inoculated soybean plants with the culture medium including propagules of the endophyte. Application of such phytohormones producing endophytes can counteract adverse effects of abiotic stresses on plant growth as shown by Khan et al. . Indeed the fungal associations have ameliorated the growth of Arabidopsis , Rapes , Solanum nigrum, Festuca arundinacea and Festuca pratensis, Lolium arundinaceum under various Cd/Ni stresses. Cu has been known to induce reduction in photosynthesis, water and nutrient uptake. Plants grown in soil containing high levels of Cu show visible symptoms of injury reflected in terms of chlorosis, growth inhibition, browning of root tips, and finally death . In addition, Cd causes decreased stomatal conductance  which affect photosynthetic rates. In present study, we observed that endophytic-fungal association activated the growth of soybean plants by improving plant biomass and synthesizing high chlorophyll, carotenoid and protein to counteract the Cu stress. The ameliorative impacts of endophyte-association were also rectified by the shoot’s carbon and hydrogen content after excursion of Cu stress. A similar effect was also observed when Neotyphodium endophytes were colonized to various grass species. Its symbiosis protected the host plants from metal toxicity of aluminum , cupper  and zinc .
In addition to plant biomass loss, excessive Cu pollution also reduces the availability and uptake of essential micronutrients in both root and shoot . Conversely in present results, the endophyte-infection increased the K, Ca and P contents in root tissues as compared to control (E-) plants under Cu stress. Besides potassium’s role in plant cellular metabolism, it plays essential part in oxidative stress responses by helping the synthesis of glutathione (GSH) which is associated with stress tolerance . In present study, the GSH level was significantly high in E + plants than E- under Cu stress. These results suggest that copper alters the equilibrium between synthesis and utilization of GSH either due to its antioxidant role or by serving as a precursor in the synthesis of phytochelatins . Similarly, our results showed that Ca content was high in the presence of endophyte during stress. Ca has an essential role in plant growth and signal transduction related to many biotic and abiotic circumstances [26, 37]. To minimize Cu toxicity and rescue plant productivity, it is essential for plants to uptake freely available mineral nutrients (such as Ca). This also indicates that increased endophytic-fungal colonization with soybean plants resulted in activation of Ca signaling to counteract stress. Previously, Kováčik et al.  indicated activation of Ca upon Cu stress. Among nutrients, phosphate is major macronutrient for plants to sustain cellular metabolism whilst availability of free phosphate can extend higher growth and metabolism in host plants [38–40]. Microorganisms are major contributor in this function. Previously, we found that P. funiculosum had high phosphate (P) solubilization potential  and hence in present study, the same helped the host soybean plants to accumulate high P as compared to endophyte-free plants during Cu stress. These ameliorative effects on soybean plants were also rectified by the reduced levels of Cu accumulation in the roots of E + plants as compared to E- plants. It further suggests that endophyte might have higher sorption of Cu during association with host-soybean plants thus accumulating low level of Cu inside roots. However, the in-depth mechanism is still unclear.
It is evident that Cu toxicity to plants also results in membrane damage due to generation of reactive oxygen species. During stress, the malondialdehyde (MDA) is generated which indicates impact on plant membrane . The significantly higher level of MDA in E- plants shows injury to functional membrane during Cu stress. This was also confirmed by the high electrolytic leakage of E- plants. However, during symbiosis with endophyte, most of the adverse effects of Cu toxicity were minimized which suggests the ameliorative function of endophyte. Excessive Cu toxicity, on the other hand, adversely affect the physiologically important free amino acid metabolism  because it lies at the crossroad between nitrogen assimilation, carbon fixation and secondary metabolism [28, 42]. Current results showed reduced amino acid metabolism in E- plants as compared to E + plants during Cu stress, which suggest rescuing role of endophyte-association to enhance cellular metabolism and improve plant growth. E + plants had significantly higher Pro, Glu, and Leu under Cu stress. Proline (Pro) has been widely known to act as osmo-protectant during abiotic stress condition whilst its accumulation in E + plants showed restoration of growth against stress. Current findings of higher nitrogen assimilation by E + plants further corroborate with the fact that even in stress conditions, the glutamate cycle and amino acid inter-conversion were not significantly affected as compared to E- plants.
Heavy metals such as Cu toxicity to the plants also increase the biosynthesis of endogenous abscisic acid (ABA) (see review of Tuteja  and reference therein). Higher ABA may also inhibit the photosynthesis rate as stress perception causes the closure of stomata to avoid dehydration. This further reduces leaf area and plant shoot length with the passage of stress period just like E- plants under stress. In present study, ABA level was significantly lower in E + plants as compared to E- plants under Cu stress. While after ten days of stress period, the E + plants still maintained higher growth as was noticed during the quantification of free amino acids, essential nutrients and ABA. Previous studies suggest that fungal inoculation can increase ABA content in leaves and roots as compared to non-inoculation control plants [43–46]. Contrarily, current findings showed that endophyte-associated soybean plants had reduced ABA as compared to control plants during stress. This altered level of ABA confirms the findings of Mauch-Mani and Mauch  and Jahromi et al., , who observed low level of ABA under stress and fungal-association. This could be due the gibberellins secretion potential of the endophytes because the non-gibberellins producing strains did not contributed towards the bioaccumulation of Cd/Cu during the screening experiment. A similar behavior of endophytes was also observed in some of the previous studies [13–15]. Sharp et al.  reported similar results with flacca mutant, however, in present study, the phosphate solubilization and gibberellins production potential of endophyte P. funiculosum has not only reprogram the plant growth but also helped the soybean plants to tolerate Cu stress.