Wheat Microbiome under Heavy Metal Stress

Healthy seeds of wheat variety Bhakkar-2000 (KJ672075) were surface sterilized with 70% ethanol for 1 min followed by 1% perchloric acid for 30 sec and rinsed thrice with ddH₂O. The seeds were then incubated to germinate for 24 h at 28 °C in petri plates. To investigate the wheat plants association with microbes under heavey metal stress, the germinated seeds from petri plates were transferd to autoclaved pots containing 3 kg soil and grown for 4 weeks in greenhouse with day light of 12 h and temperature of 22–35 °C. 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). After one week of growth, the bioactive strain (metal-resistant) grown in Czapek broth (20 ml containing 3 g of fungal mycellium) was applied to the plants according to the Khan and Lee [35 (link)]. After 15 days of growth, 80 ml of waste water collected from industrial sites of Gadun was applied to the plants for 2 weeks at 48 h of interval. The plants were finally harvested after 4 weeks for subsequent analysis. Endophyte non-inoculated wheat plants under similar conditions and setup were used as a control. To see endophytic fungal colonization potential in plant tissues, 0.5–8 mm slices of wheat roots and shoots were visualized using light microscope (Stemi SV 11 Apo, Carl Zeiss).