X-ray microanalysis of frozen-hydrated, freeze-dried, and critical point dried leaf specimens: determination of soluble and insoluble chemical elements at Erysiphe graminis epidermal cell papilla sites in barley isolines containing Ml-o and ml-o alleles

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Abstract
Three specimen preparation procedures were used in conjunction with scanning electron microscopy and energy dispersive X-ray microanalysis to determine, by comparison among preparation methods, both soluble and insoluble elements at Erysiphe graminis – barley leaf epidermal cell encounter site areas where attempted fungal penetration by appressoria failed. Near isogenic lines (RISO 5678-R and RISO 5678-S) of barley differing by mutation at the Ml-o locus were used. The recessive ml-o allele conditions barley epidermal cells to respond with papilla-associated resistance to E. graminis, while the dominant Ml-o allele allows for successful penetration of the majority of E. graminis germlings. Frozen-hydrated and freeze-dried specimens maintained soluble and insoluble elements, while specimens fixed by formalin – acetic acid – alcohol and critical point dried lost soluble elements. The effects of specimen preparation on electron-beam penetration and depth of X-ray excitation were calculated and are illustrated. Mean elemental intensity values were corrected for X-ray absorption by nickel coating of specimens (used for electrical conductivity) and for X-ray detector efficiency. The elements Cl, K, Mn, Ca, and Mg were highly soluble both at recently deposited (16 h) and at matured (24 h) papilla deposition sites. Elemental Si levels were elevated and in a partially soluble state in recently deposited papilla sites (16 h), but Si became bound or insoluble in matured (24 h) papilla sites. Elemental P and S are insoluble. The physiological role of each element is briefly discussed relative to its known function in healthy and diseased plants, with emphasis on E. graminis – barley epidermal cell encounter site penetration failure. Key words: Erysiphe graminis, Hordeum vulgare, X-ray microanalysis, scanning electron microscopy.

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